Nucleic acids encoding fungal serine protease

ABSTRACT

The present invention is related to a fungal serine protease enzyme, which comprises an amino acid sequence the mature Fa_RF7182 enzyme having an amino acid sequence of SEQ ID NO: 18. The serine protease is obtainable from  Fusarium acuminatum , more preferably from the deposited strain CBS 124084. Also disclosed are nucleic acid sequences encoding said protease, such as plasmid pALK2530 comprising the nucleotide sequence SEQ ID NO:12 deposited in  Escherichia coli  RF7803 under accession number DSM 22208 and plasmid pALK2531 comprising the full-length gene SEQ ID NO: 13 deposited in  E. coli  RF7879 under accession number DSM 22209, as well as fungal hosts, such as  Trichoderm . Said protease is useful as an enzyme preparation applicable in detergent compositions and for treating fibers, for treating wool, for treating hair, for treating leather, for treating food or feed, or for any applications involving modification, degradation or removal of proteinaceous material.

PRIORITY

This application is a divisional of U.S. application Ser. No.12/799,638, filed Apr. 28, 2010 (now U.S. Pat. No. 8,609,390), whichclaims priority from the Finnish national application number FI20095499filed on Apr. 30, 2009 and of the U.S. Provisional patent applicationNo. 61/214,974 filed on Apr. 30, 2009. The contents of all of the aboveapplications are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a fungal serine protease enzyme usefulin various applications, particularly in laundry and dish-washingdetergents. The invention relates to a nucleic acid molecule encodingsaid enzyme, a recombinant vector, a host cell for producing saidenzyme, an enzyme composition comprising said enzyme as well as aprocess for preparing such composition. This invention relates also tovarious uses of said enzyme or composition comprising said enzyme.

BACKGROUND

Microbial proteases are among the most important hydrolytic enzymes andfind applications in various industrial sectors, such as detergent,food, leather, pharmaceutical, diagnostics, waste management and silverrecovery. Microbial extracellular proteases account for a major part,more than one third, of the total worldwide industrial enzyme sales(Cherry and Fidantsef, 2003). Approximately 90% of the commercialproteases are detergent enzymes (Gupta et al., 2002). Most commercialproteases, mainly neutral and alkaline are produced by organismsbelonging to the genus Bacillus.

Serine proteases of the subtilisin family or subtilisins produced byBacillus species form the largest subgroup of industrial proteases.These enzymes are commercially important as protein degrading componentor additive of washing detergents. The commercial detergent preparationscurrently in use comprise the naturally occurring alkaline serineproteases originating or are recombinant protease preparations fromBacillus species (Maurer, 2004). Variants of the natural enzymes withimproved catalytic efficiency and/or better stability towardstemperature, oxidizing agents and changing washing conditions have beendeveloped through site-directed and/or random mutagenesis. Examples ofcommercial proteases are, such as subtilisin Carlsberg (Alcalase®,Novozymes, DK), subtilisin 309 (Savinase®, Novozymes, DK), Subtilisin147 (Esperase®, Novozymes, DK), Purafect® (Genencor Inc., USA), Kannase®(Novozymes, DK), Purafect® Ox, Properase® (Genencor Inc., USA) and theBLAP S and X series (Henkel, DE).

Several alkaline serine proteases (EC 3.4.21) and genes encoding theseenzymes have also been isolated from eukaryotic organisms, includingyeast and filamentous fungi. U.S. Pat. No. 3,652,399 and EP 519229(Takeda Chemical Industries, Ltd., JP) disclose an alkaline proteasefrom the genus Fusarium (asexual state, teleomorph) or Gibberella,(sexual state, anamorph) particularly from Fusarium sp. S-19-5 (ATCC20192, IFO 8884), F. oxysporum f. sp. lini (IFO 5880) or G. saubinetti(ATCC 20193, IFO6608), useful in the formulation of detergent and othercleanser compositions. U.S. Pat. No. 5,288,627 (NovoNordisk A/S, DK)discloses an endoprotease preparation comprising an isolated serineprotease which shows immunochemical identity to a protease derived fromF. oxysporum DSM 2672. WO 88/03946 and WO 89/04361 (Novo Industri A/S,DK) disclose an enzymatic detergent additive and a detergent compositioncomprising a protease and a lipase, wherein the fungal protease isderived from Fusarium, particularly F. oxysporum or F. solani. Adetergent additive comprising protease with specificity for peptidebonds adjacent to only one or two specific amino acids is disclosed inWO89/06270. WO1994025583 (NovoNordisk A/S, DK) discloses an activetrypsin-like protease enzyme derivable from a Fusarium species, inparticular a strain of F. oxysporum (DSM 2672), and the DNA sequenceencoding the same. The amino acid sequence of a novel protease derivingfrom Fusarium sp. BLB (FERM BP-10493) is disclosed in WO 2006101140(SODX Co. Ltd, Nakamura). Also, alkaline proteases from fungal speciessuch as Tritirachium and Conidiobolus have been reported (reviewed inAnwar and Saleemuddin, 1998).

Use of fungal serine proteases in different applications is also knownfrom several patent applications. For example, combination of acellulase and a protease, particularly a trypsin-like protease fromFusarium sp. DSM 2672 as a detergent additive or composition isdisclosed in WO 1992018599 (NovoNordisk A/S). Such detergentcompositions may further comprise reversible protease inhibitors forstabilizing the enzyme(s) as disclosed in WO 1992003529 and WO1992005239 (NovoNordisk A/S). Process for removal or bleaching ofsoiling or stains from cellulosic fabrics with an enzyme hybridcomprising a catalytically active amino acid sequence such proteaselinked to an amino acid sequence comprising a cellulose binding domainis disclosed in WO 1997028243 (NovoNordisk A/S). WO 1997002753(NovoNordisk A/S) discloses a method for gentle cleaning of soiledprocess equipment using a lipase and a protease being preferably aserine protease obtainable from Fusarium.

Due to a need to save energy the washing temperatures are decreasing. Inaddition, the current consumer demands and increased use of syntheticfibers which cannot tolerate high temperatures have changed washinghabits towards the use of low washing temperatures. EP 0290567 and EP0290569 (Novo Nordisk A/S, DK) disclose low-temperature alkalineproteases from actinomycete (Nocardiopsis dassonvillei) and fungal(Paecilomyces marquandii) micro-organisms.

The socioeconomic challenges and governmental regulations have forceddetergent industry to take in consideration many environmental aspectsincluding not only the use of more lenient chemicals, which can be usedin minor amounts and therefore leave less environmental waste trails,but also the need of energy saving. Detergent enzymes, particularlyproteases, are important ingredient in detergent compositions. The needto save energy by decreasing the washing temperatures and the increaseduse of synthetic fibers which cannot tolerate high temperatures andcurrent lifestyle have changed customer habits towards low washingtemperatures and has created a demand for new enzymes, which areeffective in low temperatures.

Despite the fact that numerous patent publications, reviews and articleshave been published, in which serine proteases from variousmicroorganisms, for example, the low temperature alkaline proteases fromactinomycete (Nocardiopsis dassonvillei) and fungal (Paecilomycesmarquandii) microorganisms are disclosed, e.g. in EP 0290567 and EP0290569 (Novo Nordisk A/S, DK), there is still a great need foralternative serine proteases, which are suitable for and effective inmodifying, degrading and removing proteinaceous materials particularlyin low or moderate temperature ranges and which are stable in thepresence of detergents with highly varying properties.

Detergent industry is making great advances in adapting its new productsto customers' habits and needs, the properties of new textile productsand new washing machines. It is evident that when developing newdetergents, particularly laundry and dish wash compositions, a widerange of varying and rapidly changing demands have to be satisfied. Inorder to fulfill all varying demands of detergent industry andgovernmental regulations, new serine protease ingredients for detergentcompositions should not only be able to accomplish their tasks in widepH and temperature ranges and remain stable in variety of conditions,including mechanical and chemical interventions in combination with avariety of different detergents, it is also desirable that the serineprotease can be produced in high amounts, which can be cost-effectivelydown-stream processed, by easy separation from fermentation broth andmycelia

SUMMARY OF THE INVENTION

The aim of the present invention is to provide a serine protease offungal origin which shows broad substrate specificity, is active atbroad pH ranges and has a broad temperature optimum, i.e. functions bothat low and moderate temperatures. The serine proteases for laundry anddish detergents have to be stable also in the presence of detergents orto be compatible with detergents. Particularly, the object of theinvention is to provide a serine protease, which is capable of removingproteinaceous material, including stains in washing laundry and dishes,at lower temperatures than the present commercial enzyme preparations,thereby saving energy. The fungal serine protease can be produced inhigh-yielding fungal hosts and its down-stream processing, e.g.separation of fermentation broth and mycelia is easy to perform.

The present invention relates to a fungal serine protease enzyme, whichhas serine protease activity and comprises an amino acid sequence of themature Fa_RF7182 enzyme as defined in SEQ ID NO:18 or an amino acidsequence having at least 81% identity to the amino acid sequence of themature Fa_RF7182 enzyme defined in SEQ ID NO:18.

The enzyme of the invention is obtainable from Fusarium acuminatum, morepreferably from the deposited strain CBS 124084.

The enzyme has a molecular mass between 25 and 35 kDa. The optimaltemperature of the enzyme is at range from 30° C. to 70° C. at pH 9.Said enzyme has pH optimum at the pH range of at least pH 6 to pH 12 at50° C. The optimal temperature and pH optimum were determined using 15min reaction time and casein as a substrate. The serine protease of theinvention is capable in degrading or removing proteinaceous stains inthe presence of detergent between 10° C. and 60° C.

The fungal serine protease enzyme of the invention is encoded by anisolated polynucleotide sequence, which hybridizes under stringentconditions with a polynucleotide sequence included in plasmid pALK2530comprising the nucleotide sequence SEQ ID No:12 deposited in E. coliRF7803 under accession number DSM 22208.

Said enzyme is encoded by an isolated polynucleotide sequence, whichencodes a polypeptide comprising an amino acid sequence of the matureFa_RF7182 enzyme as defined in SEQ ID NO:18 or an amino acid sequencehaving at least 81% identity to the amino acid sequence of the matureFa_RF7182 defined in SEQ ID NO:18. Preferably, said enzyme is encoded byan isolated nucleic acid molecule comprising the nucleotide sequence SEQID NO:17.

The full-length fungal serine protease enzyme of the invention isencoded by the polynucleotide sequence included in pALK2531 deposited inEscherichia coli RF7879 under accession number DSM 22209.

The fungal serine protease enzyme is produced from a recombinantexpression vector comprising the nucleic acid molecule encoding a fungalserine protease of the invention operably linked to regulatory sequencescapable of directing the expression of the serine protease encoding genein a suitable host. Suitable hosts include heterologous hosts,preferably microbial hosts of the genus Trichoderma, Aspergillus,Fusarium, Humicola, Chrysosporium, Neurospora, Rhizopus, Penicillium andMortiriella

Preferably said enzyme is produced in Trichoderma or Aspergillus, mostpreferably in T. reesei.

The present invention relates also to an isolated nucleic acid moleculeencoding a fungal serine protease enzyme selected from the groupconsisting of:

-   -   (a) a nucleic acid molecule encoding a polypeptide having serine        protease activity and comprising the amino acid sequence as        depicted in SEQ ID NO:18;    -   (b) a nucleic acid molecule encoding a polypeptide having serine        protease activity and has at least 81% identity to the amino        acid sequence of SEQ ID NO:18;    -   (c) a nucleic acid molecule comprising the coding sequence of        the nucleotide sequence as depicted in SEQ ID NO: 13;    -   (d) a nucleic acid molecule comprising the coding sequence of        the polynucleotide sequence contained in DSM 22208 or DSM 22209;    -   (e) a nucleic acid molecule the coding sequence of which differs        from the coding sequence of a nucleic acid molecule of any one        of (c) to (d) due to the degeneracy of the genetic code; and    -   (f) a nucleic acid molecule hybridizing under stringent        conditions with a nucleic acid molecule contained in DSM 22208,        and encoding a polypeptide having serine protease activity and        an amino acid sequence which shows at least 81% identity to the        amino acid sequence as depicted in SEQ ID NO:18.

The invention further relates to a recombinant expression vectorcomprising the nucleotide sequence of the invention operably linked toregulatory sequences capable of directing expression of said serineprotease encoding gene in a suitable host. Suitable hosts includeheterologous hosts, preferably microbial hosts of the genus Trichoderma,Aspergillus, Fusarium, Humicola, Chrysosporium, Neurospora, Rhizopus,Penicillium and Mortiriella. Preferably said enzyme is produced inTrichoderma or Aspergillus, most preferably in T. reesei.

The invention relates also to a host cell comprising the recombinantexpression vector as described above. Preferably, the host cell is amicrobial host, such as a filamentous fungus. Preferred hosts are of agenus Trichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium,Neurospora, Rhizopus, Penicillium and Mortiriella. More preferably thehost is Trichoderma or Aspergillus, most preferably a filamentous fungusT. reesei.

The present invention relates to a process of producing a polypeptidehaving serine protease activity, said process comprising the steps ofculturing the host cell of the invention and recovering the polypeptide.Also within the invention is a polypeptide having serine proteaseactivity encoded by the nucleic acid sequence of the invention and whichis obtainable by the process described above.

The invention relates to a process for obtaining an enzyme preparationcomprising the steps of culturing a host cell of the invention andeither recovering the polypeptide from the cells or separating the cellsfrom the culture medium and obtaining the supernatant. Within theinvention is also an enzyme preparation obtainable by the processdescribed above.

The invention relates to an enzyme preparation, which comprises theserine protease enzyme of the invention.

The enzyme preparation of the invention may further comprise otherenzymes selected from the group of protease, amylase, cellulase, lipase,xylanase, mannanase, cutinase, pectinase or oxidase with or without amediator as well as suitable additives selected from the group ofstabilizers, buffers, surfactants, bleaching agents, mediators,anti-corrosion agents, builders, antiredeposition agents, opticalbrighteners, caustics, abrasives, dyes, pigments and preservatives, etc.

The spent culture medium of the production host can be used as such, orthe host cells may be removed, and/or it may be concentrated, filtratedor fractionated. It may also be dried. The enzyme preparation of theinvention may be in the form of liquid, powder or granulate.

Also within the invention is the use of the serine protease enzyme orthe enzyme preparation of the invention for detergents, for treatingfibers, for treating wool, for treating hair, for treating leather, fortreating food or feed, or for any applications involving modification,degradation or removal of proteinaceous material. Particularly, theenzyme or enzyme preparation is useful as a detergent additive indetergent liquids and detergent powders.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B show the nucleotide sequence of the Fusarium acuminatumRF7182 Fa prtS8A gene (SEQ ID NO: 13) and the deduced amino acidsequence (SEQ ID NO: 14). The putative signal peptide (amino acidresidues 1-20 of SEQ ID NO:14; nucleic acid residues 1-60 of SEQ IDNO:13), analyzed by SignalP V3.0 program is in lower case letters andunderlined. The pro sequence (nucleic acid residues 61-309 and 361-429of SEQ ID NO:13) and the deduced amino acids of the pro sequence (aminoacid residues 21-126 of SEQ ID NO:14) are in lower case letters. Themature nucleotide (nucleic acid residues 430-1353 of SEQ ID NO:13) andpeptide sequences (amino acid residues 127-415 of SEQ ID NO:14) are incapital letters (N-terminal sequence determined from the purified wildtype Fa_RF7182 protein). The location of the putative intron sequence(nucleic acid residues 310-360 and 836-889 of SEQ ID NO:13) is in lowercase, italic letters and marked by a dotted line below the nucleotidesequence. The stop codon is shown by an asterisk below the sequence. TheN-terminal sequence and peptide sequences obtained from the wild typeFa_RF7182 protein are highlighted with gray background.

FIG. 1A shows the nucleotide sequence of the Fa prt8a gene (nucleotides1 to 990), the sequence region encoding amino acids Met1 to Val278 ofthe Fa_RF7182 protein.

FIG. 1B shows the nucleotide sequence of the Fa prt8A gene (nucleotides991 to 1353), the sequence region encoding amino acids Leu279 to Ala415of the Fa_RF7182 protein.

FIG. 2 schematically shows the cassette used for expressing the FaprtS8A gene in Trichoderma reesei.

FIG. 3 shows the partially purified recombinant Fa_RF7182 proteinanalysed on 12% SDS PAGE gel. Lane 1. Sample of the partially purifiedFa_RF7182, Lane 2. MW marker (Bench Mark Protein Ladder, Invitrogen).

FIG. 4A describes the temperature profile of recombinant proteinFa_RF7182 assayed at pH 9 using 15 min reaction time and casein as asubstrate. The data points are averages of three separate measurements.

FIG. 4B describes the effect of pH on the activity of recombinantFa_RF7182 protein. The buffer used was 40 mM Britton-Robinson buffer atpH 6 to pH 12 and reaction temperature was 50° C. The reaction time was15 minutes, casein was used as a substrate. The data points are averagesof three separate measurements.

FIG. 5 describes the performance of recombinant Fa_RF7182 protein withblood/milk/ink stain (Art 116, EMPA) at 30° C., pH 9, 60 min. Commercialpreparations Savinase Ultra® 16L (Novozymes A/S, DK) and Purafect® 4000L(Genencor Inc., USA) were used for comparison. ΔL* (deltaL*)=lightnessvalue L* of enzyme treated fabric−lightness value L* of fabric treatedwith buffer only (enzyme blank).

FIG. 6 describes the performance of Fa_RF7182 with blood/milk/ink stain(Art. 116, EMPA) at 50° C., pH 9, 60 min. Commercial preparationsSavinase® Ultra 16L and Purafect® 4000L were used for comparison. ΔL*(deltaL*)=lightness value L* of enzyme treated fabric−lightness value L*of fabric treated with buffer only (enzyme blank).

FIG. 7 describes the performance of recombinant Fa_RF7182 withblood/milk/ink stain (Art. 117, EMPA) in the presence of detergentpowder (Art. 601, EMPA) at 40° C. and pH 10. Commercial preparationPurafect® 4000L was used for comparison. Shown at x-axis enzyme dosage(activity/ml), at y-axis ΔL* (deltaL*)=lightness value L* of enzymetreated fabric−lightness value L* of fabric treated with buffer only(enzyme blank).

FIG. 8 shows the performance of recombinant protein Fa_RF7182 withblood/milk/ink stain (Art 117, EMPA) and liquid detergent ArielSensitive (without enzymes) at 40° C., at approximately pH 7.9, 60 min.Commercial preparation Purafect® 4000L was used for comparison. ΔL*(deltaL*)=lightness value L* of enzyme treated fabric−lightness value L*of fabric treated with buffer only (enzyme blank).

FIGS. 9A-D show the performance of recombinant protein Fa_RF7182 withblood/milk/ink stain (Art 117, EMPA) with different concentrations ofliquid base detergent for coloured fabrics at 30° C. Commercialpreparations Purafect® 4000L and Savinase® Ultra 16L were used forcomparison. ΔL* (deltaL*)=lightness value L* of enzyme treatedfabric−lightness value L* of fabric treated with buffer only (enzymeblank).

FIG. 9A shows the performance with detergent concentration of 5 g/l andpH 7.5.

FIG. 9B shows the performance with detergent concentration of 5 g/l(enzyme dosage calculated as amount of protein).

FIG. 9C shows the performance with detergent concentration of 3.3 g/land pH 7.4.

FIG. 9D shows the performance with detergent concentration of 1 g/l andpH 7.3.

FIGS. 10A-D show the performance of recombinant protein Fa_RF7182 withblood/milk/ink stain (Art 117, EMPA) with different concentrations ofAriel sensitive (without enzymes) at 30° C. Commercial preparationsPurafect® 4000L and Savinase® Ultra 16L were used for comparison. ΔL*(deltaL*)=lightness value L* of enzyme treated fabric−lightness value L*of fabric treated with buffer only (enzyme blank).

FIG. 10A shows the performance with detergent concentration of 5 g/l andpH 8.

FIG. 10B shows the performance with detergent concentration of 5 g/l(enzyme dosage calculated as amount of protein).

FIG. 10C shows the performance with detergent concentration of 3.3 g/land pH 7.9.

FIG. 10D shows the performance with detergent concentration of 1 g/l andpH 7.6.

FIGS. 11A-D show the performance of recombinant protein Fa_RF7182 ondifferent stains in Launder Ometer tests with liquid Base detergent forcoloured fabrics at 30° C. Commercial preparations Savinase® Ultra 16Land Purafect® 4000L were used for comparison. ΔL* (deltaL*)=lightnessvalue L* of enzyme treated fabric−lightness value L* of fabric treatedwith buffer only (enzyme blank).

FIG. 11A shows performance on blood/milk/ink/PE-cotton (Art. 117, EMPA).

FIG. 11B shows performance on blood/milk/ink/Cotton (Art. 116, EMPA).

FIG. 11C shows performance on grass (Art. 164, EMPA).

FIG. 11D shows performance on cocoa (Art. 112, EMPA).

FIGS. 12A and 12B describe total stain removal efficiency (delta % SR)of Fa_RF7182 enzyme preparation on eight different protease sensitivestains (Table 5) in full-scale washing trials. Commercial preparationsSavinase® Ultra 16L and Purafect® 4000L were used for comparison.

FIG. 12A shows total stain removal efficiency when protease preparationswere dosed according to the activity.

FIG. 12B shows total stain removal efficiency when protease preparationswere dosed according to the amount of protein

FIGS. 13A-E describe stain removal effect with liquid detergent base forcolored fabrics in full scale trial at 30° C.

FIG. 13A shows the stain removal effect on chocolate milk/pigment/Cotton(C-03-030/CFT).

FIG. 13B shows the stain removal effect on blood/milk/ink/Cotton(C-05-059b/CFT).

FIG. 13C shows the stain removal effect on blood/milk/ink/PE-Cotton(C-05-014/CFT).

FIG. 13D shows the stain removal effect on groundnut oil/milk/Cotton(C-05-014/CFT).

FIG. 13E shows the stain removal effect on egg Yolk/Pigment/Cotton(CS-38-010/CFT).

FIGS. 14A-F describe the performance of recombinant Fa_RF7182 proteinwith blood/milk/ink stain (Art 117, EMPA) at temperatures from 10° C. to60° C., pH 9, 60 min. Commercial preparations Savinase Ultra® 16L(Novozymes A/S, DK), Purafect® 4000L (Genencor Inc, USA) and Properase®4000E (Genencor Inc., USA) were used for comparison. ΔL*(deltaL*)=lightness value L* of enzyme treated fabric−lightness value L*of fabric treated with buffer only (enzyme blank).

FIG. 14A shows the performance of recombinant protein Fa_RF7182 andcommercial protease preparations at 10° C.

FIG. 14B shows the performance of recombinant protein Fa_RF7182 andcommercial protease preparations at 20° C.

FIG. 14C shows the performance of recombinant protein Fa_RF7182 andcommercial protease preparations at 30° C.

FIG. 14D shows the performance of recombinant protein Fa_RF7182 andcommercial protease preparations at 40° C.

FIG. 14E shows the performance of recombinant protein Fa_RF7182 andcommercial protease preparations at 50° C.

FIG. 14F shows the performance of recombinant protein Fa_RF7182 andcommercial protease preparations at 60° C.

FIGS. 15A and 15B show the performance of recombinant Fa_RF7182 proteinwith blood/milk/ink stain (Art 117, EMPA) and Liquid Base concentrationof 3.3 g/l at 10° C. and 20° C. Commercial preparations Savinase® Ultra16L, Purafect® 4000L and Properase® 4000 E were used for comparison. ΔL*(deltaL*)=lightness value L* of enzyme treated fabric−lightness value L*of fabric treated with buffer only (enzyme blank).

FIG. 15A Shows performance at 10° C.

FIG. 15B Shows performance at 20° C.

SEQUENCE LISTING

SEQ ID NO:1 Sequence of an amino terminal peptide #3811 from Fusariumacuminatum RF7182 protease.

SEQ ID NO:2 Sequence of a tryptic peptide 1609,880 from Fusariumacuminatum RF7182 protease.

SEQ ID NO:3 Sequence of a tryptic peptide 1793,957 from Fusariumacuminatum RF7182 protease.

SEQ ID NO:4 Sequence of a tryptic peptide 743,494 from Fusariumacuminatum RF7182 protease.

SEQ ID NO:5 Sequence of a tryptic peptide 2103,832 (start 2045,06) fromFusarium acuminatum RF7182 protease.

SEQ ID NO:6 Sequence of a tryptic peptide 2103,832 (start 1406,57) fromFusarium acuminatum RF7182 protease.

SEQ ID NO:7 Sequence of a tryptic peptide 3662,692 from Fusariumacuminatum RF7182 protease.

SEQ ID NO:8 The sequence of the oligonucleotide primer PRO123 derivedfrom the peptide SEQ ID NO:3.

SEQ ID NO:9 The sequence of the oligonucleotide primer PRO122 derivedfrom the peptide SEQ ID NO:6.

SEQ ID NO:10 The sequence of the serine protease consensusoligonucleotide primer PRO60.

SEQ ID NO:11 The sequence of the serine protease consensusoligonucleotide primer PRO61.

SEQ ID NO:12 The sequence of the PCR fragment obtained using the primersPRO123 (SEQ ID NO:8) and PRO61 (SEQ ID NO:11) and Fusarium acuminatumRF7182 genomic DNA as a template.

SEQ ID NO:13 The nucleotide sequence of the full-length Fusariumacuminatum RF7182 protease gene (Fa prtS8A).

SEQ ID NO:14 The deduced amino acid sequence of the full-length Fusariumacuminatum RF7182 protease (Fa_RF7182) including amino acids Met 1 toAla415.

SEQ ID NO:15 The nucleotide sequence encoding the amino acid sequence ofthe proenzyme form of Fusarium acuminatum RF7182 protease.

SEQ ID NO:16 The amino acid sequence of the proenzyme form of Fusariumacuminatum RF7182 protease, including amino acids Ala21 to Ala415 of thefull-length Fa_RF7182 defined in SEQ ID No:14.

SEQ ID NO:17 The nucleotide sequence encoding the amino acid sequence ofthe mature form of Fusarium acuminatum RF7182 protease.

SEQ ID NO:18 The amino acid sequence of the mature form of Fusariumacuminatum RF7182 protease, including amino acids Ala127 to Ala415 ofthe full-length Fa_RF7182 defined in SEQ ID No:14.

Depositions

Fusarium acuminatum RF7182 was deposited at the Centraalbureau VoorSchimmelcultures at Uppsalalaan 8, 3508 AD, Utrecht, the Netherlands on28 Jan. 2009 and assigned accession number CBS 124084.

The E. coli strain RF7803 including the plasmid pALK2530 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany on 21 Jan.2009 and assigned accession number DSM 22208.

The E. coli strain RF7879 including the plasmid pALK2531 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7 b, D-38124 Braunschweig, Germany on 21 Jan.2009 and assigned accession number DSM 22209.

DETAILED DESCRIPTION

The present invention provides a serine protease of fungal origin, whichprotease shows broad substrate specificity, is stable at high pH rangesand has a broad temperature optimum, i.e. good performance both at lowand moderate temperatures. The enzyme is ideal for detergentapplications, withstanding oxidizing and chelating agents and beingeffective at low enzyme levels in detergent solutions. Particularly, theserine protease is active at temperatures as low as 10° C., thepreferred range being from 10° C. to 70° C. Thus, the present inventionprovides an alternative serine protease for use in detergent and otherapplications. The fungal serine protease can be produced inhigh-yielding fungal hosts and its down-stream processing, e.g.separation of fermentation broth and mycelia is easy to perform.

By “serine protease” or “serine endopeptidase” or “serineendoproteinase” is in connection to this invention meant an enzymeclassified as EC 3.4.21 by the Nomenclature of the International Unionof Biochemistry and Molecular Biology. Serine proteases are found inboth single-cell and complex organisms. Based on their structuralsimilarities, serine proteases have been grouped into at least six clans(SA, SB, SC, SE, SF and SG; S denoting serine protease), which have beenfurther subgrouped into families with similar amino acid sequences andthree-dimensional structures (see, for example the Serine protease homepage at biochem.wustl.edu/˜protease/, Department of Biochemistry andMolecular Biophysics, Washington University of Medicine, St. Louis, Mo.,USA). These protein hydrolyzing or degrading enzymes are characterizedby the presence of a nucleophilic serine group in their active site, andthe proteases of clan SA and clan SB are also distinguished by havingessential aspartate and histidine residues, which along with the serine,form a catalytic triad.

The major clans include the “chymotrypsin-like”, including chymotrypsin,trypsin and elastase (clan SA) and “subtilisins-like” (clan SB) serineproteases. The enzymes target different regions of the polypeptidechain, based upon the side chains of the amino acid residues surroundingthe site of cleavage. The serine protease of the present inventionbelongs to clan SB.

The characterized “subtilisin-like serine proteases” or “subtilases” aregenerally bacterial in origin. This class of proteases, represented byvarious Bacillus, like B. amyloliquifaciens, B. licheniformis and B.subtilis (Rao et al., 1998), is specific for aromatic or hydrophobicresidues, such as tyrosine, phenylalanine and leucine.

By the term “serine protease activity” as used in the invention is meanthydrolytic activity on protein containing substrates, e.g. such ascasein, haemoglobin, keratin and BSA. The methods for analysingproteolytic activity are well-known in the literature and are referrede.g. in Gupta et al. (2002).

Proteases can be classified using group specific inhibitors. The diversegroup of “serine protease inhibitors”, includes synthetic chemicalinhibitors and natural proteinaceous inhibitors. One group of naturalinhibitors are serpins (abbreviated from serine protease inhibitors),such as antithrombin and alpha 1-antitrypsin. Artificial syntheticinhibitors include 3,4-dichloroisocoumarin (3,4-DCI),diisopropylfluorophosphate (DFP), phenylmethylsulfonyl fluoride (PMSF)and tosyl-L-lysine chloromethyl ketone (TLCK). Some of the serineproteases are inhibited by thiol reagents such asp-chloromercuribenzoate (PCMB) due to the presence of a cysteine residuenear the active site. Thus, the serine protease activity can bedetermined in an assay based on cleavage of a specific substrate or inan assay using any protein containing substrate with or without aspecific inhibitor of serine proteases under suitable conditions.

Serine proteases are generally active at neutral or alkaline pH, with anoptimum between pH 7 and 11, and have broad substrate specificity. The“alkaline serine proteases” mean enzymes that are active and stable atpH 9 to pH 11 or even at pH 10 to 12.5 (Shimogaki et al., 1991) and haveisoelectric point around pH 9. Those represent the largest subgroup ofcommercial serine proteases. The molecular masses of alkaline serineproteases range between 15 and 35 kDa. The temperature optima of thenatural serine proteases are around 60° C. (Rao et al., 1998).

Microorganism strains capable of producing protease activity can bescreened and the activity on different substrates can be determined.Chosen strains can be cultivated on a suitable medium. After asufficient amount of an interesting serine protease has been produced,the enzyme can be isolated or purified and its properties can be morethoroughly characterized. Alternatively, genes encoding serine proteasesin various organisms can be isolated and the amino acid sequence encodedby the genes can be compared with the amino acid sequences of the serineprotease isolated and characterized in the Examples here.

The produced protease enzymes, particularly the serine proteases can bepurified by using conventional methods of enzyme chemistry, such as saltpreparation, ultrafiltration, ion exchange chromatography, affinitychromatography, gel filtration and hydrophobic interactionchromatography. Purification can be monitored by protein determination,enzyme activity assays and by SDS polyacrylamide gel electrophoresis.The enzyme activity and stability of the purified enzyme at varioustemperature and pH values as well as the molecular mass and theisoelectric point can be determined.

The purification of a preferred serine protease of the present inventionhas been demonstrated in Example 1b. The filtrated culture supernatantwas applied to a Q Sepharose FF column. The flow through fraction wasapplied to phenyl Sepharose HP column and proteins were eluted with alinear decreasing salt gradient. Fractions showing protease activitywere pooled, concentrated and applied to a Superdex 75 10/300 GL column.Purification was followed by activity assays on resorufin-labeled caseinas described in Example 1b. Naturally, it is possible to separate theenzyme of the present invention by using other known purificationmethods instead, or in addition to the methods described herein. Therecombinant serine protease was purified as described in Example 5 andused for characterization of pH and temperature profiles.

The molecular mass of the purified serine protease can be determined bymass spectrometry or on SDS-PAGE according to Laemmli (1970). Themolecular mass can also be predicted from the amino acid sequence of theenzyme. The mature serine protease or mature serine protease enzymetypically has a molecular mass between 20 to 35 kDa, typically around 25to 30 kDa (Rao et al., 1998).

The serine proteases are synthesized as inactive “zymogenic precursors”or “zymogens” in the form of a preproenzyme, which are activated byremoval of the signal sequence (secretion signal peptide or prepeptide)and the prosequence (propeptide) to yield an active mature form of theenzyme (Chen and Inouye, 2008). This activation process involves actionof proteases and may result from limited self-digestive or autocatalyticprocessing of the serine protease. The prosequence may be cleaved forexample during posttranslational phases of the production or in thespent culture medium or during the storage of the culture medium orenzyme preparation. Activation of the proenzyme may also be achieved byadding a proteolytic enzyme capable of converting the inactive proenzymeinto active mature enzyme into the culture medium where the hostorganism is cultivated or adding the proteolytic enzyme to the culturesupernatant after cultivation process. The shortening of the enzyme canalso be achieved e.g. by truncating the gene encoding the polypeptideprior to transforming it to the production host.

The term “mature” means the form of enzyme which after removal of thesignal sequence and propeptide comprises the essential amino acids forenzymatic or catalytic activity. In filamentous fungi it is the nativeform secreted into the culture medium.

The temperature optimum of the serine protease can be determined in asuitable buffer at different temperatures by using casein as a substrateas described in Example 1c and 5 or by using other substrates and buffersystems described in the literature (Gupta et al., 2002). Determinationof the pH optimum can be carried out in a suitable buffer at differentpH values by following the activity on a protein substrate.

Protease activity is generally based on degradation of solublesubstrates. In detergent application proteases have to work onsubstances which are at least partly insoluble. Thus an importantparameter for a detergent protease is its the ability to adsorb to andhydrolyse these insoluble fragments.

Another important parameter for selection of detergent proteases is itsisoelectric point or pI value. The detergent proteases perform best whenthe pH value of the detergent solution in which it works isapproximately the same as the pI value for the enzyme. The pH in thedetergent application is usually alkaline, pH 7-9 for liquid detergentsand pH 9-10.5 when powder detergents are used. pI can be determined byisoelectric focusing on an immobilized pH gradient gel composed ofpolyacrylamide, starch or agarose or by estimating the pI from the aminoacid sequence, for example by using the pI/MW tool at ExPASy server(expasy.org/tools/pi_tool.html; Gasteiger et al., 2003).

The N-terminus of the purified protease as well as internal peptides canbe sequenced according to Edman degradation chemistry (Edman and Begg,1967) as described in Example 2 or by other methods described in theliterature.

The serine protease enzyme of the invention may derive from any organismincluding bacteria, archaea, fungi, yeasts and even higher eukaryote,such as plants. Preferably said enzyme originates from a fungus,including filamentous fungi and yeasts, for example from a genusselected from the group comprising Fusarium. Fungal alkaline proteasesare advantageous to the bacterial proteases due to the ease ofdown-stream processing to produce a microbe-free enzyme or enzymecomposition. Mycelium can be easily removed through filtrationtechniques prior to the purification of the enzyme.

The present invention relates to fungal serine protease, which has agood performance in the presence of detergents with highly varyingproperties, at broad, i.e. from low to moderate temperature ranges, suchas 10° C. to 60° C.

In the present invention good performance in presence of detergent meansthat the enzyme, in this case the fungal serine protease of theinvention, functions at lower temperature ranges than many commercialsubtilisins presently for sale. In other words, good performance meansthat the enzyme is capable of degrading or removing proteinaceous stainsor material at low to moderate temperature ranges, but especially atlower temperature ranges than the present commercial products, forexample the commercial enzyme product Purafect® 4000L (Genencor Inc.,USA).

The fungal serine protease of the invention functions at low temperatureranges. For example, by modifying pH, selecting detergents with suitableproperties, including enzyme protecting agents and by controllingwashing conditions the activity of the serine protease of the inventionmay be maintained at temperatures as low as 10° C. Therefore, the serineprotease of the invention depending on the washing conditions andauxiliary ingredients and additives in detergents is useful particularlyin temperatures at or below 50° C. The enzyme functions also attemperatures at or below 45° C., at or below 40° C., at or below 35° C.,or at or below 30° C.

In the presence of a detergent, the fungal serine protease of theinvention functions as defined above between 10° C. and 60° C. InExamples 6 to 13, comparative experiments are described, and from FIGS.7 to 15 it is evident that the performance of the fungal serine proteaseFa_RF7182 in varying conditions and exposed to varying treatments, onmultitude of different stains on different textile material, measured asdeltaL* or Sum of delta % SR, is by far better than the performance ofthe commercial products, Savinase® Ultra 16L (Novozymes A/S, DK),Purafect® 4000L (Genencor Inc, USA) and Properase® 4000E (Genencor Inc.,USA). Particularly, the stain removal effect of said fungal serineprotease Fa_RF7182 in low temperature ranges such as from 10° C. to 40°C. is remarkably higher than with Savinase® Ultra 16L, Properase® 4000Eand Purafect® 4000L.

From said experimental results it can be concluded that the fungalserine protease of the invention is capable of satisfying the greatlyvarying demands of detergent customers and detergent industry andindustry providing washing machinery and is well suited to therequirements of future regulations and customer habits.

According to a preferred embodiment of the invention the fungal serineprotease enzyme is a polypeptide having serine protease activity andcomprising the mature enzyme of Fa_RF7182 having the amino acid sequenceof SEQ ID NO:18 or an amino acid sequence having at least 81% identityto the amino acid sequence SEQ ID NO:18 or at least 80% to the aminoacid sequence SEQ ID NO:14. Preferred enzymes show at least 81%,preferably at least 83%, more preferably at least 85%, even morepreferably at least 87% identity. Still more preferably the amino acidsequences show at least 89% or at least 91%, 92%, 93%, 95% or 97%, morepreferably at least 98%, most preferably 99% identity to the amino acidsequence of SEQ ID NO:18. The identities of the two enzymes are comparedwithin the corresponding sequence regions, e.g. within the full-lengthor mature region of the serine protease.

The serine protease of the present invention is marked Fa_RF7182, anisolated serine protease originating from Fusarium acuminatum and is amember of clan SB, family 8 of serine endoproteinases.

By the term “identity” is here meant the identity between two amino acidsequences compared to each other within the corresponding sequenceregion having approximately the same amount of amino acids. For example,the identity of a full-length or a mature sequence of the two amino acidsequences may be compared. The amino acid sequences of the two moleculesto be compared may differ in one or more positions, which however doesnot alter the biological function or structure of the molecules. Suchvariation may occur naturally because of different host organisms ormutations in the amino acid sequence or they may be achieved by specificmutagenesis. The variation may result from deletion, substitution,insertion, addition or combination of one or more positions in the aminoacid sequence. The identity of the sequences is measured by usingClustalW alignment (e.g. in ebi.ac.uk/Tools/Clustalw). The matrix usedis as follows: BLOSUM, Gap open: 10, Gap extension: 0.5.

Preferably, the fungal serine protease is obtainable from Fusarium, morepreferably from Fusarium acuminatum. According to the most preferredembodiment the serine protease of the invention is obtainable from thestrain deposited at Centraalbureau voor Schimmelcultures under accessionnumber CBS 124084.

One preferred embodiment of the invention is a fungal serine proteaseenzyme having serine protease activity and an amino acid sequence of themature Fa_RF7182 enzyme as defined in SEQ ID NO:18. The mature enzymelacks the signal sequence or prepeptide and the prosequence orpropeptide. The mature serine protease of the invention includes aminoacids Ala127 to Ala415 of the full length protease characterized in SEQID NO:13. Thus, within the scope of the invention is also thefull-length Fa_RF7182 enzyme having SEQ ID NO:13 including the signalsequence (prepeptide) and propeptide and the mature enzyme as well asthe proenzyme form lacking the signal sequence (prepeptide) thus havingSEQ ID NO:15.

The present invention relates to a fungal serine protease enzyme, themature form of which has a molecular mass or molecular weight between 20and 35 kDa, preferably between 25 and 33 kDa, more preferably between 28and 30 kDa. The most preferred MW is the predicted molecular mass ofFa_RF7182 being 29 kDa for the mature polypeptide obtained by using theCompute pI/MW tool at ExPASy server (Gasteiger et al., 2003).

The enzyme of the invention is effective in degrading proteinaceousmaterial at a broad temperature range. The optimal temperature of theenzyme is from 30° C. to 70° C. (about 10% of the maximum activity),preferably from 30° C. to 60° C. (approximately 30% of the maximumactivity), and more preferably between 40° C. and 50° C. (at least 60%of the maximum activity), most preferably at 50° C. (the maximumactivity, Fa_RF7182) when measured at pH 9 using 15 min reaction timeand casein as a substrate as described in Example 5.

According to one preferred embodiment of the invention the fungal serineprotease enzyme has pH optimum at pH range from at least pH 6 to pH 12,showing at least 10% of the maximum activity at pH 7 at 50° C. using 15min reaction time as described in Example 5. Preferably, the enzyme haspH optimum at pH range of pH 6 to pH 11 (at least 40% of the maximumactivity). In particular, the pH optimum is between pH 6 and pH 10(approximately 55% of the maximum activity), and more preferably betweenpH 6 and pH 9 (at least 80% of the maximum activity), and mostpreferably at pH 7 at 50° C. using 15 min reaction time as described inExample 5 when using casein as a substrate.

The fungal serine protease of the invention has “good performance in thepresence of detergent”, i.e. is capable of degrading or removingproteinaceous stains on colored materials in the presence of detergentat low temperature ranges, specifically at lower temperature ranges thanthe present commercial products, for example the commercial enzymeproduct Purafect® 4000L (Genencor Inc., USA). In the presence of adetergent the enzyme of the invention functions between 10° C. and 60°C., preferably between 20° C. and 50° C., more preferably at or below50° C. The Fa_RF7182 enzyme functions also at temperatures at or below45° C., at or below 40° C., at or below 35° C., or at or below 30° C.

The serine protease enzyme of the invention has pI, which as predictedfrom the deduced amino acid sequence is between pI 8.8 and pI 9.5,preferably between pI 8.9 and pI 9.3. The predicted pI of Fa_RF7182enzyme of the invention is pI 9.1.

Oligonucleotides synthesized on the amino acid sequence of N-terminal ortryptic peptides of the purified enzyme or a PCR product obtained byusing the above oligonucleotides can be used as probes in isolation ofcDNA or a genomic gene encoding the serine protease of the invention.The probe may be designed also based on the known nucleotide or aminoacid sequences of homologous serine proteases. The serine proteaseclones may also be screened based on activity on plates containing aspecific substrate for the enzyme or by using antibodies specific for aserine protease.

According to a preferred embodiment of the invention the fungal serineprotease enzyme is encoded by an isolated polynucleotide sequence whichhybridizes under stringent conditions with a polynucleotide or probesequence included in plasmid pALK2530 comprising the nucleotide sequenceSEQ ID NO:12 in E. coli RF7803, deposited at the Deutsche Sammlung vonMikroorganismen and Zellkulturen (DSMZ) under accession number DSM22208.

In the present invention the Fa prt8A gene was isolated with a probeprepared by PCR using stringent hybridization as described in Example3d. Standard molecular biology methods can be used in isolation of cDNAor a genomic DNA of the host organism, e.g. the methods described in themolecular biology handbooks, such as Sambrook and Russell, 2001.

Hybridization with a DNA probe, such as for example SEQ ID NO:12consisting of more than 100-200 nucleotides, is usually performed at“high stringency” conditions, i.e. hybridization at a temperature, whichis 20-25° C. below the calculated melting temperature (Tm) of a perfecthybrid, the Tm calculated according to Bolton and McCarthy (1962).Usually prehybridization and hybridization are performed at least at 65°C. in 6×SSC (or 6×SSPE), 5×Denhardt's reagent, 0.5% (w/v) SDS, 100 μg/mldenatured, fragmented salmon sperm DNA. Addition of 50% formamide lowersthe prehybridization and hybridization temperatures to 42° C. Washes areperformed in low salt concentration, e.g. in 2×SSC-0.5% SDS (w/v) for 15minutes at room temperature (RT), followed in 2×SSC-0.1% SDS (w/v) atRT, and finally in 0.1×SSC-0.1% SDS (w/v) at least at 65° C.

According to one preferred embodiment the fungal serine protease enzymeof the invention is encoded by an isolated nucleic acid molecule, whichencodes a polypeptide comprising the amino acid sequence characterizedin SEQ ID NO:18, or a polypeptide having at least 81% to the amino acidsequence SEQ ID NO:18 or at least 80% to the amino acid sequence SEQ IDNO:13. Preferred enzymes show at least 81%, preferably at least 83%,more preferably at least 85%, even more preferably at least 87%identity. Still more preferably the amino acid sequences show at least89%, 91%, 93% or at least 97%, more preferably at least 98%, mostpreferably 99% identity to the amino acid sequence of SEQ ID NO:18. Theidentities of the two enzymes are compared within the correspondingsequence regions, e.g. within the mature or full-length region of theserine protease.

Thus, within the scope of the invention is a polypeptide sequence, whichis encoded by a nucleic acid molecule encoding the amino acid sequenceof the full-length serine protease of the invention including theprepeptide (signal sequence) and the propeptide in addition to themature form of the enzyme, and which amino acid sequence ischaracterized in SEQ ID NO:14.

Also, within the scope of the invention is a polypeptide sequence, whichis encoded by a nucleic acid molecule encoding the proenzyme form ofserine protease enzyme of the invention including the propeptide inaddition to the mature form of the enzyme, and which amino acid sequenceis characterized in SEQ ID NO:16.

One preferred embodiment of the invention is the fungal serine proteaseenzyme encoded by an isolated nucleic acid molecule, which comprises thenucleotide sequence encoding the mature form of the Fa_RF7182 serineprotease having SEQ ID NO:18.

According to one preferred embodiment the fungal serine protease enzymeof the invention is encoded by an isolated nucleic acid moleculecomprising the nucleotide sequence SEQ ID NO:17 encoding the mature formof the Fa_RF7182 enzyme (SEQ ID NO:18).

Thus, within the scope of the invention is the polypeptide encoded bythe nucleic acid molecule having the nucleotide sequence SEQ ID NO:13comprising the “coding sequence” for the enzyme. The expression “codingsequence” means the nucleotide sequence which initiates from thetranslation start codon (ATG) and stops at the translation stop codon(TAA, TAG or TGA). The translated full-length polypeptide starts usuallywith methionine and comprises intron regions.

Also, within the scope of the invention is a fungal serine proteaseenzyme encoded by a nucleic acid molecule comprising the nucleotidesequence SEQ ID NO:15, which encodes the Fa_RF7182 proenzyme form.

According to another preferred embodiment of the invention the fungalserine protease is encoded by the polynucleotide sequence included inpALK2531 deposited in E. coli RF7879 under accession number DSM 22209.

One embodiment of the invention is the serine protease enzyme producedfrom a recombinant expression vector comprising the nucleic acidmolecule, which encodes the fungal serine protease enzyme ascharacterized above operably linked to regulatory sequences capable ofdirecting the expression of said serine protease encoding gene in asuitable host. Construction of said recombinant expression vector anduse of said vector is described in more detail in Example 4.

Suitable hosts for production of the fungal serine protease enzyme arehomologous or heterologous hosts, such as the microbial hosts includingbacteria, yeasts and fungi. Filamentous fungi, such as Trichoderma,Aspergillus, Fusarium, Humicola, Chrysosporium Neurospora, Rhizopus,Penicillium and Mortiriella are preferred production hosts due to theease of down-stream processing and recovery of the enzyme product.Suitable hosts include species such as T. reesei, A. niger, A oryzae, A.sojae, A. awamori or A. japonicus type of strains, F. venenatum or F.oxysporum, H. insolens or H. lanuginosa, N. crassa and C. lucknowense,some of which are listed as enzyme production host organisms in e.g.AMFEP 2007 list of commercial enzymes (http://www.amfep.org/list.html).More preferably, the enzyme is produced in a filamentous fungal host ofthe genus Trichoderma or Aspergillus, such as T. reesei, or A. niger, A.oryzae or A. awamori strains. According the most preferred embodiment ofthe invention the fungal serine protease enzyme is produced in T.reesei.

The present invention relates also to an isolated nucleic acid moleculeencoding the fungal serine protease enzyme selected from the groupconsisting of:

-   -   (a) a nucleic acid molecule encoding a polypeptide having serine        protease activity and comprising the amino acid sequence as        depicted in SEQ ID NO:18;    -   (b) a nucleic acid molecule encoding a polypeptide having serine        protease activity and at least 81% to SEQ ID NO:18;    -   (c) a nucleic acid molecule comprising the coding sequence of        the nucleotide sequence as depicted in SEQ ID NO: 13;    -   (d) a nucleic acid molecule comprising the coding sequence of        the polynucleotide sequence contained in DSM 22208 or DSM 22209;    -   (e) a nucleic acid molecule the coding sequence of which differs        from the coding sequence of a nucleic acid molecule of any one        of (c) to (d) due to the degeneracy of the genetic code; and        -   (f) a nucleic acid molecule hybridizing under stringent            conditions with a nucleic acid molecule contained in DSM            22208, and encoding a polypeptide having serine protease            activity and an amino acid sequence which shows at least 81%            identity to the amino acid sequence as depicted in SEQ ID            NO:18.

The nucleic acid molecule of the invention may be RNA or DNA, whereinthe DNA may constitute of the genomic DNA or cDNA.

Standard molecular biology methods can be used in isolation and enzymetreatments of the polynucleotide sequence encoding the fungal serineprotease of the invention, including isolation of genomic and plasmidDNA, digestion of DNA to produce DNA fragments, sequencing, E. colitransformations etc. The basic methods are described in the standardmolecular biology handbooks, e.g. Sambrook and Russell, 2001.

Isolation of the Fa prtS8A gene encoding the Fa_RF7182 polypeptide isdescribed in Example 3. Briefly, the 753 bp PCR fragment obtained byusing the sequences of the degenerate oligonucleotide primers PRO123(SEQ ID NO: 8) and PRO61 (SEQ ID NO:11) was used to isolate the Fa prt8Agene from Fusarium acuminatum RF7182 in pBluescript 11 KS+ vector. Thefull-length Fusarium acuminatum Fa prtS8A gene was included in theplasmid pALK2531 deposited in E. coli to the DSMZ culture collectionunder accession number DSM 22209. The deduced amino acid sequence of theserine protease was analysed from the DNA sequence.

The nucleotide sequence of Fusarium acuminatum serine protease Fa prtS8Anucleotide sequence (SEQ ID NO: 13) and the deduced sequence (SEQ ID NO:14) are presented in FIGS. 1A and 1B. The length of the gene is 1353 bp(including the stop codon). Two putative introns were found having thelength of 51 bps and 54 bps. The deduced protein sequence consists of415 amino acids including a predicted signal sequence of 20 amino acids(SignalP V3.0; Nielsen et al., 1997 and Nielsen and Krogh, 1998) and apropeptide from Ala21 to Arg 126. The predicted molecular mass was 29kDa for the mature polypeptide and the predicted pI was 9.1. Thesepredictions were made using the Compute pI/MW tool at ExPASy server(Gasteiger et al., 2003). The deduced amino acid sequence contained twopossible N-glycosylation sites (Asn79 and Asn258), but according to CBSServer NetNGlyc V1.0 only the site at position Asn79 (located in thepro-peptide) is probable. The homologies to the published proteasesequences were searched using the BLASTX program, version 2.2.9 at NCBI(National Center for Biotechnology Information) (Altschul et al., 1990).The identity values of the mature Fa_RF7182 sequence to thecorresponding mature regions of homologous sequences were obtained byusing ClustalW alignment (Matrix: BLOSUM, Gap open: 10, Gap extension:0.5 (e.g. in ebi.ac.uk/Tools/Clustalw) and are shown in Table 3.

The serine protease Fa-RF7182 of the present invention showed highesthomology to Gibberella zeae (Fusarium graminearum) hypothetical proteinPH-1, locus tag FG03315.1 (EMBL accession no. XP-383491, unpublished),to Hypocrea lixii (Trichoderma harzianum) CECT 2413 serine endopeptidase(EMBL accession no. CAL25580, Suarez et al., 2007) and to T. atroviridealkaline proteinase precursor S08.066, ALP (EMBL accession no. M87516,Geremia et al. 1993), the latter disclosed as an amino acid sequence SEQID NO:313 in U.S. 60/818,910 (Catalyst Bioscience Inc.). The identity toG. zeae hypothetical protein was within the full-length enzyme 79%. Whenthe mature polypeptides lacking the signal sequence and propeptide werealigned, the identity was 80%. Identity to H. lixii CECT 2413 serineendopeptidase was 73% (full-length enzyme) and 79% (mature enzyme). Theidentity to T. atroviride ALP was 71% (full-length enzyme) and 76%(mature enzyme).

Thus, within the scope of the invention is an isolated polynucleotidesequence or isolated nucleic acid molecule, which encodes a fungalserine protease enzyme or polypeptide comprising the amino acid sequenceof the mature form of the Fa_RF7182 enzyme characterized in SEQ ID NO:18, i.e. amino acids Ala127 to Ala415 of the full length serine proteaseof SEQ ID NO:14.

Further, within the scope of the present invention are nucleic acidmolecules which encode a fragment of a fungal serine proteasepolypeptide, wherein the fragment has serine protease activity and hasat least 81% identity to the amino acid sequence SEQ ID NO:18 or atleast 80% to the amino acid sequence SEQ ID NO:14. Preferred enzymesshow at least 81%, preferably at least 83%, more preferably at least85%, even more preferably at least 87% identity. Still more preferablythe amino acid sequences show at least 89% or at least 91%, 92%, 93%,95% or 97%, more preferably at least 98%, most preferably 99% identityto the amino acid sequence of SEQ ID NO:18. The identities of the twoenzymes are compared within the corresponding sequence regions, e.g.within the mature or full-length region of the serine protease.

The nucleic acid molecule is preferably a molecule comprising the codingsequence as depicted in SEQ ID NO:13, which encodes the full length formof the fungal serine protease enzyme of this invention.

The isolated nucleic acid molecule of the invention may be a moleculecomprising the coding sequence of the polynucleotide sequence containedin DSM 22208 or DSM 22209. DSM 22208 carries the nucleotide sequence ofthe PCR fragment (SEQ ID NO:12) used in cloning the full length FaprtS8A gene. DSM 22209 carries the nucleotide sequence of the fulllength Fa prtS8A gene (SEQ ID NO:13).

The nucleic acid molecule of the invention may also be an analogue ofthe nucleotide sequence characterized in above. The “degeneracy” meansanalogues of the nucleotide sequence, which differ in one or morenucleotides or codons, but which encode the recombinant protease of theinvention.

The nucleic acid molecule may also be a nucleic acid moleculehybridizing under stringent conditions to a PCR probe contained inplasmid pALK2530 deposited in E. coli under the accession number DSM22208 and encoding a polypeptide having serine protease activity and anamino acid sequence which within the corresponding sequence region showsat least 81% identity to the amino acid sequence as depicted in SEQ IDNO:18. The hybridizing DNA may originate from a fungus belonging tospecies Fusarium or it may originate from other fungal species.

Thus, within the scope of the invention is an isolated nucleic acidmolecule comprising a nucleotide sequence as depicted in SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:15 or SEQ ID NO:17 and analogues thereof.

The present invention relates also to a recombinant expression vector orrecombinant expression construct, which can be used to propagate orexpress the nucleic acid sequence encoding the chosen serine protease ina suitable prokaryotic or eukaryotic host. The recombinant expressionvector comprises DNA or nucleic acid sequences which facilitate ordirect expression and secretion of the serine protease encoding sequencein a suitable host, such as promoters, enhancers, terminators (includingtranscription and translation termination signals) and signal sequencesoperably linked the polynucleotide sequence encoding said serineprotease. The expression vector may further comprise marker genes forselection of the transformant strains or the selection marker may beintroduced to the host in another vector construct by co-transformation.Said regulatory sequences may be homologous or heterologous to theproduction organism or they may originate from the organism, from whichthe gene encoding the serine protease is isolated.

Examples of promoters for expressing the serine protease of theinvention in filamentous fungal hosts are the promoters of A. oryzaeTAKA amylase, alkaline protease ALP and triose phosphate isomerase,Rhizopus miehei lipase, Aspergillus niger or A. awamori glucoamylase(glaA), Fusarium oxysporum trypsin-like protease, Chrysosporiumlucknowense cellobiohydrolase I promoter, Trichoderma reeseicellobiohydrolase I (Cel7A) etc.

In yeast, for example promoters of S. cerevisiae enolase (ENO-1),galactokinase (GAL1), alcohol dehydrogenase (ADH2) and3-phosphoglycerate kinase can be used to provide expression.

Examples of promoter sequences for directing the transcription of theserine protease of the invention in a bacterial host are the promoter oflac operon of Escherichia coli, the Streptomyces coelicolor agarase dagApromoter, the promoter of the B. licheniformis alpha-amylase gene(amyL), the promoter of the B. stearothermophilus maltogenic amylasegene (amyM), the promoters of the B. sublitis xylA and xylB genes, etc.

Suitable terminators include those of the above mentioned genes or anyother characterized terminator sequences.

Suitable transformation or selection markers include those whichcomplement a defect in the host, for example the dal genes from B.subtilis or B. licheniformis or Aspergillus amdS and niaD. The selectionmay be based also on a marker conferring antibiotic resistance, such asampicillin, kanamycin, chloramphenicol, tetracycline, phleomycin orhygromycin resistance.

Extracellular secretion of the serine protease of the invention ispreferable. Thus, the recombinant vector comprises sequencesfacilitating secretion in the selected host. The signal sequence of theserine protease of the invention or the presequence or prepeptide may beincluded in the recombinant expression vector or the natural signalsequence may be replaced with another signal sequence capable offacilitating secretion in the selected host. Thus, the chosen signalsequence may be homologous or heterologous to the expression host.

Examples of suitable signal sequences are those of the fungal or yeastorganisms, e.g. signal sequences from well expressed genes, well knownin art.

The recombinant vector may further comprise sequences facilitatingintegration of the vector into the host chromosomal DNA to obtain stableexpression.

The Fa_RF7182 protease of the invention was expressed with its ownsignal sequence from the T. reesei cbh1 (cel7A) promoter as described inExample 4. The expression construct used to transform the T. reesei hostincluded also cbh1 terminator and amdS marker for selecting thetransformants from the untransformed cells.

The present invention relates also to host cells comprising therecombinant expression vector as described above. Suitable hosts forproduction of the fungal serine protease enzyme are homologous orheterologous hosts, such as the microbial hosts including bacteria,yeasts and fungi. Production systems in plant or mammalian cells arealso possible.

Filamentous fungi, such Trichoderma, Aspergillus, Fusarium, Humicola,Chrysosporium, Neurospora, Rhizopus, Penicillium and Mortiriella, arepreferred production hosts due to the ease of down-stream processing andrecovery of the enzyme product. Suitable expression and production hostsystems are for example the production system developed for thefilamentous fungus host Trichoderma reesei (EP 244234), or Aspergillusproduction systems, such as A. oryzae or A. niger (WO 9708325, U.S. Pat.No. 5,843,745, U.S. Pat. No. 5,770,418), A. awamori, A. sojae and A.japonicus-type strains, or the production system developed for Fusarium,such as F. oxysporum (Malardier et al., 1989) or F. venenatum, and forNeurospora. crassa, Rhizopus miehei, Mortiriella alpinis, H. lanuginosaor H. insolens or for Chrysosporium lucknowensee (U.S. Pat. No.6,573,086). Suitable production systems developed for yeasts are systemsdeveloped for Saccharomyces, Schizosaccharomyces or Pichia pastoris.Suitable production systems developed for bacteria are a productionsystem developed for Bacillus, for example for B. subtilis, B.licheniformis, B. amyloliquelaciens, for E. coli, or for theactinomycete Streptomyces. Preferably the serine protease of theinvention is produced in a filamentous fungal host of the genusTrichoderma or Aspergillus, such as T. reesei, or A. niger, A oryzae, A.sojae, A. awamori or A. japonicus-type strains. According the mostpreferred embodiment of the invention the fungal serine protease enzymeis produced in T. reesei.

The production host cell may be homologous or heterologous to the serineprotease of the invention. The host may be free of homogenous proteasesdue to removal of proteases either by inactivation or removal of one ormore host proteases, e.g. by deletion of the gene(s) encoding suchhomogenous or homologous proteases.

The present invention relates also to a process for producing apolypeptide having serine protease activity, said process comprising thesteps of culturing the natural or recombinant host cell carrying therecombinant expression vector for a serine protease of the inventionunder suitable conditions and optionally isolating said enzyme. Theproduction medium may be a medium suitable for growing the host organismand containing inducers for efficient expression. Suitable media arewell-known from the literature.

The invention relates to a polypeptide having serine protease activity,said polypeptide being encoded by the nucleic acid molecule of theinvention and which is obtainable by the process described above.Preferably, the polypeptide is a recombinant protease enzyme obtained byculturing the host cell carrying the recombinant expression vector for aserine protease enzyme of the invention.

The invention further relates to a process for obtaining an enzymepreparation comprising a polypeptide, which has serine proteaseactivity, said process comprising the steps of culturing a host cellcarrying the expression vector of the invention and either recoveringthe polypeptide from the cells or separating the cells from the culturemedium and obtaining the supernatant having serine protease activity.

The present invention relates also to an enzyme preparation, whichcomprises the serine protease enzyme characterized above. The enzymepreparation or composition has serine protease activity and isobtainable by the process according to the invention.

Within the invention is an enzyme preparation which comprises the fungalserine protease of the invention, preferably the recombinant serineprotease obtained by culturing a host cell, which carries therecombinant expression vector of the invention.

Said enzyme preparation may further comprise different types of enzymesin addition to the serine protease of this invention, for exampleanother protease, an amylase, a lipase, a cellulase, cutinase, apectinase, a mannanase, a xylanase and/or an oxidase such as a laccaseor peroxidase with or without a mediator. These enzymes are expected toenhance the performance of the serine proteases of the invention byremoving the carbohydrates and oils or fats present in the material tobe handled. Said enzymes may be natural or recombinant enzymes producedby the host strain or may be added to the culture supernatant after theproduction process.

Said enzyme preparation may further comprise a suitable additiveselected from the group of surfactants or surface active agent, buffers,anti-corrosion agents, stabilizers, bleaching agents, mediators,builders, caustics, abrasives, optical brighteners, antiredepositionagents, dyes, pigments and preservatives, etc.

Surfactants are useful in emulsifying grease and wetting surfaces. Thesurfactant may be a non-ionic including semi-polar and/or anionic and/orcationic and/or zwitterionic.

Buffers may be added to the enzyme preparation to modify pH or affectperformance or stability of other ingredients.

Suitable stabilizers include polyol such as propylene glycol orglycerol, a sugar or sugar alcohol, lactic acid, boric acid, or boricacid derivatives, peptides, etc.

Bleaching agent is used to oxidize and degrade organic compounds.Examples of suitable chemical bleaching systems are H₂O₂ sources, suchas perborate or percarbonate with or without peracid-forming bleachactivators such as tetraacetylethylenediamine, or alternativelyperoxyacids, e.g. amide, imide or sulfone type. Chemical oxidizers maybe replaced partially or completely by using oxidizing enzymes, such aslaccases or peroxidases. Many laccases do not function effectively inthe absence of mediators.

Builders or complexing agents include substances, such as zeolite,diphosphate, triphosphate, carbonate, citrate, etc. The enzymepreparation may further comprise one or more polymers, such ascarboxymethylcellulose, poly(ethylene glycol), poly(vinyl alcohol),poly(vinylpyrrolidone), etc. Also, softeners, caustics, preservativesfor preventing spoilage of other ingredients, abrasives and substancesmodifying the foaming and viscosity properties can be added.

According to one preferred embodiment of the invention said enzymepreparation is in the form of liquid, powder or granulate.

The fungal serine protease of the present invention may like otherproteases, particularly alkaline proteases be used in the detergent,protein, brewing, meat, photographic, leather, dairy and pharmaceuticalindustries (Kalisz, 1988, Rao et al., 1998). For example, it may be usedas an alternative to chemicals to convert fibrous protein waste (e.g.horn, feather, nails and hair) to useful biomass, protein concentrate oramino acids (Anwar and Saleemuddin, 1998). The use of fungal serineprotease of the present invention may like other enzymes provesuccessful in improving leather quality and in reducing environmentalpollution and saving energy and it may like alkaline proteases be usefulin resolution of the mixture of D,L-amino acids. Subtilisin incombination with broad-spectrum antibiotics in the treatment of burnsand wounds is an example of the use of serine proteases inpharmaceutical industry, therefore the fungal serine protease of thepresent invention may also find such use and may also like alkalineproteases be applicable in removal of blood on surgical equipments andcleaning contact lenses or dentures. Like alkaline protease fromConidiobolus coronatus, the fungal serine protease of the presentinvention may be used for replacing trypsin in animal cell cultures. Theproteases of the invention can also be used in cleaning of membranes anddestruction of biofilms. In baking proteases are used e.g. indestruction of the gluten network to produce non-elastic doughs withvery low water retention. In other food applications, the food proteinsare hydrolysed using proteases with the aim of e.g. solubilisingprotein, rendering (extracting more protein from animal bones), creatingnew flavours, reducing bitterness, changing emulsifying properties,generating bioactive peptides and reducing allergenicity of proteins,treating yeast or milk. The substrates include animal, plant andmicrobial proteins.

Detergent industry, particularly the laundry detergent industry, hasemerged as the single major consumer of proteases active at high pHrange (Anwar and Saleemuddin, 1998). The ideal detergent protease shouldpossess broad substrate specificity to facilitate the removal of largevariety of stains due to food, grass, blood and other body secretions.It has to be active in the pH and ionic strength of the detergentsolution, the washing temperature and pH, and tolerate mechanicalhandling as well as the chelating and oxidizing agents added todetergents. The pI of the protease must be near the pH of the detergentsolution. Due to present energy crisis and the awareness for energyconservation, it is currently desirable to use the protease at lowertemperatures.

The present invention relates also to the use of the serine proteaseenzyme or the enzyme preparation for detergents, treating textilefibers, for treating wool, for treating hair, for treating leather, fortreating feed or food, or for any application involving modification,degradation or removal of proteinaceous material.

One preferred embodiment of the invention is therefore the use of theserine protease enzyme as characterized above as a detergent additiveuseful for laundry detergent and dish wash compositions, includingautomatic dish washing compositions.

The expression “detergent” is used to mean substance or materialintended to assist cleaning or having cleaning properties. The term“detergency” indicates presence or degree of cleaning property. Thedegree of cleaning property can be tested on different proteinaceous orprotein containing substrate materials or stains or stain mixtures boundto solid, water-insoluble carrier, such as textile fibers or glass.Typical proteinaceous material includes blood, milk, ink, egg, grass andsauces. For testing purposes mixtures of proteinaceous stains arecommercially available. The function of the detergent enzyme is todegrade and remove the protein-containing stains. Test results depend onthe type of stain, the composition of the detergent and the nature andstatus of textiles used in the washing test (Maurer, 2004).

Typically, protease or wash performance is measured as “stain removalefficiency” or “stain removal effect” or “degree of cleaning property”,meaning a visible and measurable increase of lightness or change incolour of the stained material, e.g. in artificially soiled swatches ortest cloths. Lightness or change in colour values can be measured, forexample by measuring the colour as reflectance values with aspectrophotometer using L*a*b* colour space coordinates as described inExamples 6 to 10. Fading or removal of proteinaceous stain indicating ofthe protease performance (stain removal efficiency) is calculated forexample as ΔL*, which means lightness value L* of enzyme treated fabricminus lightness value L* of fabric treated with buffer or washing liquorwithout enzyme (enzyme blank or control). The presence of detergent mayimprove the performance of the enzyme in removing the stains.

The serine protease of the present invention degrades various kinds ofproteinaceous stains under conditions of neutral and alkaline pH andeven in the presence detergents with different compositions (as shown inExamples 6 to 13).

As shown in Example 6 the serine protease of the invention removed theblood/milk/ink standard stain at 50° C. and especially at 30° C. in pH 9buffer and preservatives better than the commercial proteasepreparations Savinase® Ultra 16L and Purafect® 4000L (FIGS. 5 and 6).The enzyme preparations were dosed as activity units. The stain removaleffect on blood/milk/ink stain was tested also at the whole temperaturerange from 10° C. to 60° C. as described in Example 12. Fa_RF7182protease preparation showed higher stain removal capacity compared tothe commercial protease preparation Savinase® Ultra 16L, Properase®4000E and Purafect® 4000L especially at low washing temperatures, at arange from 10° C. to 40° C. (FIG. 14).

The performance of the Fa_RF7182 protease was tested also in detergentpowder at 40° C. at pH 10 as described in Example 7. The ability of theenzyme in removing blood/milk/ink standard stain on polyester-cottonmaterial (Art.117, EMPA) in presence of phosphate containing referencedetergent was determined. Each enzyme preparation was dosed as activityunits (μmol tyrosine/minute). As shown in FIG. 7 the protease of theinvention is suitable also for powder detergents at very alkalineconditions. The performance of Fa_RF7182 enzyme was similar tocommercial protease Purafect® 4000L at 40° C.

The Fa_RF7182 protease removed blood/milk/ink standard stain onpolyester-cotton material (Art.117, EMPA) also in the presence of liquiddetergent Ariel Sensitive without enzymes (Procter & Gamble, UK) asshown in Example 8. The enzyme was dosed as activity units (μmoltyrosine/minute/volume). The dosage of 4-8 units of commercial enzymepreparations per ml of liquor was equal to dosage of about 0.2-0.5% ofenzyme preparation per weight of detergent, which is typical level foruse of detergent enzymes. The Fa_RF7182 showed excellent performancewith liquid detergent (FIG. 8) and the increase in lightness of thestained material was considerably higher than with the commercialpreparations Savinase® Ultra 14L and Purafect® 4000L.

Similar results were observed when the performance of the Fa_RF7182enzyme was tested using different liquid detergent concentrations(Example 9, FIGS. 9A-D and 10A-D) at 30° C. The efficiency of Fa_RF7182on blood/milk/ink stain was considerably higher compared to commercialpreparations Purafect® 4000L and Savinase® Ultra 14 L with bothdetergents and at all detergent concentrations, when same amount ofactivity was dosed. Also if dosing was calculated as amount of addedprotein (FIGS. 9B and 10B), the stain removal efficiency was the highestwith Fa_RF7182. Washings were performed also at lower temperatures witha liquid detergent concentration of 3.3 g/l as described in Example 13.At 10° C. and 20° C. the Fa_RF7182 preparation showed superiorperformance over the commercial preparations Savinase® Ultra 14L,Purafect® 4000L and Properase® 4000E (FIG. 15).

In addition to the blood/milk/ink stains on cotton or polyester cottonfabrics, the Fa_RF7182 serine protease was effective in removing otherstains, such as grass (Art.164, EMPA) and cocoa (Art.112, EMPA) whentested in liquid detergents at 30° C. Treatments were performed in ATLASLP-2 Launder-Ometer. Results (FIG. 11) show that the Fa_RF7182 waseffective in removal of different stains at low temperatures like 30° C.

The performance of recombinant Fa_RF7182 enzyme preparation produced inT. reesei was tested in the presence of liquid base detergent in fullscale in a washing machine at 30° C. (Example 11) and compared tocommercial preparations Savinase® Ultra 16L and Purafect® 4000L. Eightdifferent protease sensitive tracers for testing side effects arepresented in Table 5 and the process conditions in Table 6. Enzymedosages used in the test trials were calculated both as enzymeactivities and as amount of enzyme protein. Results with the differentstains presented in FIGS. 12 and 13 show that Fa_RF7182 protease wasmore effective than the commercial enzyme preparations. The Fa_RF7182was most efficient on blood/milk/ink, chocolate/milk, groundnut oil/milkand egg yolk (FIG. 13).

As observed from FIGS. 5 to 11 the lightness L* of the test material wasremarkably increased in the presence of the fungal serine protease ofthe invention. Using Fa_RF7182 enzyme preparation, better lightnesscompared to competitor products was obtained with similar enzymeactivity dosage or in other words, similar lightness compared tocompetitor products was obtained with lower enzyme activity dosage.

The Fa_RF7182 protease was also tested in full-scale trials at 30° C.using liquid detergent and a selection of protease sensitive tracers(Example 11), In these tests, the total stain removal effect, based onthe sum of the results obtained with the eight different proteasesensitive stains (Stains 1-8, Table 5) was considerably higher withFa_RF7182 compared to commercial protease preparations Purafect® 4000Land Savinase® Ultra 16L, when proteases were dosed as amount of activity(FIG. 12A). Fa_RF7182 was efficient especially on blood/milk/ink,chocolate/milk, groundnut oil/milk and egg yolk (FIGS. 13A-E).

According to a preferred embodiment of the invention the fungal serineprotease of the invention is useful in detergent liquids and detergentpowders as shown in Examples 6 to 11. The enzyme or enzyme preparationof the invention may be formulated for use in a hand or machine laundryor may be formulated for use in household hard surface cleaning orpreferably in hand or machine dish washing operations.

Example 1 Production and Purification of the Fusarium acuminatum RF7182Protease

(a) Cultivation of Fusarium acuminatum RF7182 Protease

Fungal strain originally named as TUB F-2008 was isolated from a soilsample as a filamentous fungus growing on alkaline (pH 8.5) agaroseplate. It produced protease activity, according to hydrolysis of caseinand haemoglobin included in the agar plate. As the plate cultivationswere performed at 8-10° C. this result suggested that TUB F-2008produces a protease or proteases acting at cold temperatures. TUB F-2008was identified as Fusarium acuminatum Ellis & Everh (identified byArthur de Cock at Identification Services, Centralbureau VoorSchimmelcultures, P.O. Box 85167, 3508 AD Utrecht, The Netherlands) andwas renamed as RF7182. The F. acuminatum RF7182 was grown, maintainedand sporulated on Potato Dextrose (PD) agar (Difco) at +4° C. For enzymeproduction spores from RF7182 slant were inoculated into a culturemedium which contained: 30 g/l Corn meal (finely ground), 5 g/l Cornsteep powder, 4 g/l Soybean meal (defatted), 2 g/l KH₂PO₄, 1 g/l NaCland 1 g/l Paraffin oil. The pH of the medium was adjusted beforesterilization with NaOH to 8.5 and the medium was autoclaved for 30minutes at 121° C. The microbe was cultivated in 50 ml volume on ashaker (200 rpm) at 28° C. for 7 days. The spent culture supernatant wasshown to contain alkaline protease activity, when activity measurementswere performed (according to Example 1c) at different pH values (pH7-10). Because of this alkaline activity, the RF7182 strain was chosenas putative protease gene donor strain.

(b) Purification of Protease from the F. acuminatum RF7182 CultureMedium

Cells and solids were removed from the spent culture medium bycentrifugation for 30 min, 50000 g at +4° C. (Sorvall RC6 plus). 50 mlof the supernatant was used for purification of the protease. Aftercentrifugation, pH of supernatant was adjusted to 8.0 with addition ofHCl. The supernatant was then filtered through 0.44 μm filter (MILLEX HVMillipore) and applied to a 5 mL Q Sepharose FF column (GE Healthcare)equilibrated in 20 mM Tris-HCl, pH 8. Flow through fraction wascollected and its pH was lowered to 7.5 by adding HCl. Solid ammoniumsulfate was added to the flow through fraction to obtain a final saltconcentration of 1 M. The flow through fraction was then filteredthrough 0.44 μm filter before applying to phenyl Sepharose HP (5 mL)column (GE Healthcare) equilibrated in 20 mM Tris-HCl-1M ammoniumsulfate pH 7.5. The proteins were eluted with a linear decreasingammonium sulfate gradient (from 1 to 0 M). Fractions of 5 ml werecollected and analyzed for protease activity on resorufin-labeled casein(Boehringer Mannheim Biochemica) at pH 8.0 as instructed by themanufacturer. Fractions with protease activity were pooled and ultrafiltrated with 10 k membrane (Amicon). The concentrated filtrate wasapplied to a Superdex 75 10/300 GL column (GE Healthcare) equilibratedwith 20 mM Tris-HCl-200 mM NaCl, pH 7.5. Proteins were eluted with thesame buffer and 0.5 ml fractions were collected. The protease activityfrom these fractions was analysed. The fractions with protease activitywere analyzed on sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE). The fractions were shown to contain onemajor protein band having a molecular mass of about 29 kDa. The chosenfractions were pooled. The pooled fractions were used for preparation ofpeptides (Example 2). This purified F. acuminatum RF7182 protease wasnamed as Fa_RF7182.

(c) Protease Activity Assay

Protease activity was assayed by the casein Folin-Ciocalteau methodusing casein as a substrate. Rate of casein degradation by a proteasewas measured by spectrophotometrical monitoring of the release ofacid-soluble fragments as a function of time. Casein substrate used inthe assay was prepared as follows: 6 g of Casein Hammerstein Grade MPBiomedicals, LLC (101289) was dissolved in 500 ml of 30 mM Tris, 2.0 mMCaCl₂, 0.7 mM MgCl₂, 2.5 mM NaHCO₃. The pH of the substrate solution wasadjusted to 8.5. The enzyme reactions were stopped using 0.11 M TCAsolution. The Folin reagent used in the assay was prepared by diluting25 ml of 2 N Folin-Ciocalteu's phenol reagent (SIGMA, F 9252) to 100 mlby distilled water. The reaction was started by first incubating 2.5 mlof substrate solution for 5 min at 50° C. after which 0.5 ml of enzymesolution was added and reaction was conducted for 30 min. After 30 minreaction 2.5 ml of reaction stop solution was added, the contents weremixed and allowed to stand for 30 minutes at room temperature. Tubeswere centrifuged 4000 rpm for 10 minutes (Hettich Rotanta 460). One mlof clear supernatant was mixed with 2.5 ml 0.5 M Na₂CO₃ and 0.5 mldiluted Folin reagent. After waiting for at least 5 min (colordevelopment) the absorbance of the mixture (colour) was measured at 660nm against an enzyme blank. The enzyme blank was prepared as follows:0.5 ml enzyme solution was mixed with 2.5 ml stopping solution and 2.5ml substrate, and the mixture was incubated for 30 min at 50° C. Oneunit of enzyme activity was defined as the enzyme quantity thatliberates the acid soluble protein hydrolysis product corresponding to 1μg of tyrosine per ml (or g) of the reaction mixture per min.

Example 2 N-Terminal and Internal Amino Acid Sequencing of the PurifiedFusarium acuminatum RF7182 Protease

For determination of internal sequences, the Coomassie Brilliant Bluestained band was cut out of the polyacrylamide gel and “in-gel” digestedessentially as described by Shevchenko et al. (1996). Proteins werereduced with dithiothreitol and alkylated with iodoacetamide beforedigestion with trypsin (Sequencing Grade Modified Trypsin, V5111,Promega).

Electrospray ionization quadrupole time-of-flight tandem mass spectrafor de novo sequencing were generated using a Q-TOF instrument(Micromass, Manchester, UK) connected to an Ultimate nano liquidchromatograph (LC-Packings, The Netherlands) essentially as describedpreviously (Poutanen et al., 2001) but using a 150 μm×1.0 mm trappingcolumn (3 μm, 120A, #222403, SGE Ltd UK) for peptide preconcentration.

For N-terminal sequence analysis SDS-PAGE/separated proteins weretransferred by electroblotting into a polyvinylidine difluororidemembrane (ProBlott; Perkin Elmer Applied Biosystems Division, FosterCity, Calif.) After being stained with Coomassie brilliant blue, theprotein bands of interest were removed and subjected to N-terminalsequence analysis by Edman degradation on a Procise 494A proteinsequencer (Perkin Elmer Applied Biosystems Division, Foster City,Calif.)

The N-terminal and internal peptide sequences determined from thepurified Fa_RF7182 protease are shown in Table 1. The peptide sequencesshowed homology to a published hypothetical protease sequence fromGibberella zeae (Fusarium graminearum) with EMBL Accession numberXP_(—)383491. According to comparison of the sequences, all the peptidesequences obtained were located close to the N-terminus of the matureFa_RF7182 protein.

TABLE 1 N-terminal and internal peptide sequences determined from Fusariumacuminatum RF7182 protease Fa_RF7182. Peptide Sequence SEQ ID NOComments #3811 A(L/I)TXQSNAPW SEQ ID NO: 1 N-terminal sequence 1609, 880SGAPWG(L/I)Q(L/I)SHK SEQ ID NO: 2 1793, 957 (A/L/I)(A/L/I)TTQSGAPWGSEQ ID NO: 3 (L/I)GA(L/I)SHK 743, 494 (A/N)(A/N)(L/I)(L/I)SVKSEQ ID NO: 4 2103, 832 GGAHTD SEQ ID NO: 5 (start 2045, 06) 2103, 832NGHGTHVAGT(L/I)NTK SEQ ID NO: 6 (start 1406, 57) 3662, 692TSY(L/I)YDTTAGSGSYGYYVDSG(L/I) SEQ ID NO: 7 N(L/I)AHYS(L/I)NR

Example 3 Cloning of the Fusarium acuminatum RF7182 Gene EncodingFa_RF7182 Protein

(a) Isolation of DNA and Molecular Biology Methods Used

Standard molecular biology methods were used in the isolation and enzymetreatments of DNA (e.g. isolation of plasmid DNA, digestion of DNA toproduce DNA fragments), in E. coli transformations, sequencing etc. Thebasic methods used were either as described by the enzyme, reagent orkit manufacturer or as described in the standard molecular biologyhandbooks, e.g. Sambrook and Russell (2001). Isolation of genomic DNAfrom F. acuminatum RF7182 was done as described in detail by Raeder andBroda (1985).

(b) Primers for Probe Preparation

The probe for cloning the gene encoding the Fa_RF7182 protein wasamplified by PCR. Degenerate sense oligonucleotides were planned basingon the amino acid sequences of the peptides obtained from the purifiedRF7182 (Table 1) and antisense oligonuclotides using a chosen consensusarea from different published S8 type serine protease sequences.Conserved sequence areas were determined from alignment of sequenceswith accession numbers AAR11460 (Trichophyton rubrum), CAD24010(Microsporum canis), AAT65816 (Penicillium nordicum), Q5NDC0(Penicillium chrysogenum) and AAC27316 (Fusarium oxysporum). Thecombinations of the primers in the PCR reactions were chosen accordingto the location of the peptide homologues in the published proteasesequences. The sequences of the primers are shown in Table 2 (SEQ IDNOs: 8-11).

TABLE 2  The oligonucleotides (SEQ ID NOs: 8-11) synthesized as PCRprimers in probe amplification. Oligos, SEQ ID NOs, oligolengths and degenaracies, oligonucleotides and the amino acidsof the peptide used in planning of the oligonucleotide sequence. SEQ IDLength Oligo NO: (nts) Degeneracy Sequence^((a) Peptide^((b) PRO123 8 201024 CARWCNGGNGCNCCNTGGGG (s) 1793, 957 PRO122 9 20 1024CAYGGNACNCAYGTNGCNGG (s) 2103, 832 (start 1406, 57) PRO60 10 22 64GMRGCCATRGAGGTWCCRGTSA (as) PRO61 11 20 32 GGMGMRGCCATRGAGGTWCC (as)^((a)R = A or G, S = C or G, W = A or T, M = A or C, Y = T or C, N = A,C, T or G; “s” in the parenthesis = sense strand, “as” in theparenthesis = antisense strand. ^((b)The peptides sequences are includedin Table 1.c) PCR Reactions and Selection of Probes for Cloning

F. acuminatum RF7182 genomic DNA was used as template for probesynthesis. The PCR reaction mixtures contained 1× Phusion™ GC Buffer,0.25 mM dNTPs, 5% DMSO, 1 μM each primer and 2 units of Phusion™ DNApolymerase (Finnzymes, Finland) and approximately 3 μg of genomic DNAper 100 μl reaction volume. The conditions for the PCR reactions werethe following: 1 min initial denaturation at 98° C., followed by 29cycles of 10 sek at 98° C., 30 sek annealing at 48-58.5° C., 30 sekextension at 72° C. and a final extension at 72° C. for 5 min. Primercombination PRO123 (SEQ ID NO:8) and PRO61 (SEQ ID NO:11) produced aspecific DNA product having the expected size (calculated from thepublished fungal protease sequences). The DNA product was isolated andpurified from the PCR reaction mixture and cloned to pCR® 4-TOPO® vectoraccording to the manufacturers instructions (Invitrogen, USA). The 753bp PCR fragment was sequenced from this plasmid (SEQ ID NO: 12). ThepCR® 4-TOPO® plasmid containing this PCR amplified DNA fragment wasnamed pALK2530. The E. coli strain RF7803 including the plasmid pALK2530was deposited to the DSM collection under the accession number DSM22208.

The deduced amino acid sequence of the PCR fragment included thesequences of the internal RF7182 peptides SEQ ID NOs:1-7. Peptides withSEQ ID NOs: 6 and 7 and the C-terminal part of the peptide SEQ ID NO:2matched partly to the deduced amino acid sequence. Peptides with SEQ IDNO:4 and SEQ ID NO:5 and the C-terminal part of the peptide SEQ ID NO: 3matched completely (Table 1). This confirms that the DNA fragmentobtained from the PCR reaction was part of the gene encoding theFa_RF7182 protein and was thus used as a probe for screening the fulllength gene from F. acuminatum RF7182 genomic DNA.

(d) Cloning of the F. acuminatum RF7182 Gene Encoding Fa_RF7182 Protease

F. acuminatum genomic DNA was digested with several restriction enzymesfor Southern blot analysis. The hybridization was performed with the 771kb EcoRI fragment including SEQ ID NO:12 from the plasmid pALK2530 as aprobe (Example 3c). The above probe was labeled by using digoxigeninaccording to supplier's instructions (Roche, Germany). Hybridization wasperformed over night at 65° C. After hybridization the filters werewashed 2×5 min at RT using 2×SSC-0.1% SDS followed by 2×15 min at 65° C.using 0.1×SSC-0.1% SDS.

Several hybridizing fragments in the F. acuminatum RF7182 genomic DNAdigests could be detected. Genomic SalI digests contained a hybridizingfragment of about 5 kb in size. The single hybridizing fragment size waslarge enough to contain the full length gene encoding the RF7182 proteinaccording to calculations basing on published fungal protease sequences.Genomic DNA fragments were isolated from the RF7182 genomic SalI digestfrom the size range of the hybridizing fragment. The genomic fragmentsisolated from agarose gel were cloned to pBluescript II KS+(Stratagene,USA) vectors cleaved with SalI. Ligation mixture was transformed toEscherichia coli XL10-Gold cells (Stratagene) and plated on LB(Luria-Bertani) plates containing 50-100 μg/ml ampicillin. The E. colicolonies were screened for positive clones using colonial hybridizationwith the pALK2530 insert as a probe. Hybridization was performed asdescribed for the RF7182 genomic DNA digests. Several clearly positiveclones were collected from the plates and one was confirmed withsequencing. The full-length gene encoding the Fa_RF7182 protease (SEQ IDNO:13) was sequenced from the 5 kb SalI insert and the plasmidcontaining this insert was named pALK2531. The E. coli strain RF7879including the plasmid pALK2531 was deposited to the DSM collection underthe accession number DSM 22209. The gene encoding the Fa_RF7182 proteasewas named as Fa prtS8A.

(e) Characterisation of the Gene Encoding Fa_RF7182 Protease and theDeduced Amino Acid Sequence

The Fa prtS8A sequence (SEQ ID NO:13) and the deduced amino acidsequence (SEQ ID NO:14) are shown in FIGS. 1A and 1B. The length of thegene is 1353 bp (including the stop codon). Two putative introns werefound having the lengths of 51 bps and 54 bps (5′ and 3′ bordersequences according to those of fungal introns, according to Gun et al.,1987). The deduced protein sequence (SEQ ID NO:14) consists of 415 aminoacids including a predicted signal sequence of 20 amino acids (SignalPV3.0; Nielsen et al., 1997 and Nielsen and Krogh, 1998). The N-terminalparts of peptides with SEQ ID NOs:1-3, not included in the probesequence, were included in the deduced amino acid sequence. The peptidewith SEQ ID NO:3 matched the deduced amino acid sequence completely. Thepredicted molecular mass was 28568.64 Da for the mature polypeptide andthe predicted pI was 9.08. These predictions were made using the ComputepI/MW tool at ExPASy server (Gasteiger et al., 2003). The deduced aminoacid sequence contained two possible N-glycosylation sites at amino acidpositions Asn79 and Asn258 (FIG. 1), but according to CBS ServerNetNGlyc V1.0 only the site at position Asn79 (located in thepro-peptide) is probable.

(f) Homology, Identity and Alignment Studies

The homologies to the published protease sequences were searched usingthe BLASTX program version 2.2.9 at NCBI (National Center forBiotechnology Information) with default settings (Altschul et al.,1990). The highest homologies were to a hypothetical protein fromGibberella zeae (Fusarium graminearum) (EMBL accession number XP 383491)and Hypocrea lixii (Trichoderma harzianum) serine endopeptidase (EMBLAccession number CAL25580). Also homology was found to a sequenceincluded in the patent application U.S. 60/818,910 (Catalyst BiosciencesInc.), SEQ ID 313 in the application. The RF7182 mature amino acidsequence was aligned with the above homologous mature amino acidsequences. The identity values obtained by using ClustalW alignment(ebi.ac.uk/Tools/Clustalw; Matrix: BLOSUM, Gap open: 10, Gap extension:0.5, EMBL-EBI) are shown in Table 3.

TABLE 3 The identity values (%) obtained from ClustalW alignment of thededuced Fa_RF7182 protease amino acid sequences. The mature amino acidsequences excluding the signal peptides and propeptides were aligned.Matrix: BLOSUM, Gap open: 10, Gap extension: 0.5, EMBL_EBI. G. zeae,XP_383491; T. harzianum, CAL25508; US 60/818,910, SEQ ID NO: 313 in theapplication. Fa_RF7182 G. zeae T. harz. US 60/818,910 Fa_RF7182 100 8079 76 G. zeae 100 78 76 T. harz. 100 94 US 60/818,910 100

Example 4 Production of the Recombinant Fa_RF7182 in Trichoderma reesei

(a) Preparing the Production (Host) Vector

The expression plasmid pALK2536 was constructed for production ofrecombinant RF7182 protease in Trichoderma reesei. The Fa prtS8A genewith its own signal sequence was exactly fused to the T. reesei cbh1(cel7A) promoter by PCR. The Fa prtS8A gene fragment was excised fromits 3′-end by BamHI (a site created after stop codon in PCR). Thisleaves no original Fa prtS8A terminator in the construct prior to thecbh1 terminator sequence. An amdS marker gene was added to theconstruction including the cbh1 promoter, the Fa prtS8A gene and cbh1terminator. The construction is analogous to that described in Paloheimoet al. (2003) and the 8.7 kb linear expression cassette is presented inFIG. 2. The expression cassette was isolated from the vector backboneafter EcoRI digestion and was used for transforming T. reeseiprotoplasts. The host strain used does not produce any of the four majorT. reesei cellulases (CBHI, CBHII, EGI, EGII). The transformations wereperformed as in Penttilä et al. (1987) with the modifications describedin Karhunen et al. (1993). The transformants were purified on selectionplates through single conidia prior to sporulating them on PD.

(b) Protease Production in Shake Flasks and Laboratory Scale Bioreactor

The transformants were inoculated from the PD slants to shake flaskscontaining 50 ml of complex lactose-based cellulase inducing medium(Joutsjoki et al., 1993) buffered with 5% KH₂PO₄ at pH 6.0. The proteaseproduction of the transformants was analyzed from the culturesupernatants after growing them for 7 days at 30° C., 250 rpm. InSDS-PAGE gels, a major protein band of about 29 kDa corresponding torecombinant Fa_RF7182 protease was detected from the spent culturesupernatants. The protease activity was assayed using casein as asubstrate as described in Example 1c. Clearly increased activitiescompared to host were measured from the culture supernatants. Theintegration of the expression cassette into the fungal genomes wasconfirmed from chosen transformants by using Southern blot analysis inwhich several genomic digests were included and the expression cassettewas used as a probe.

The T. reesei transformants producing the best protease activities inthe shake flask cultivations were chosen to be cultivation in laboratoryscale bioreactors. Cellulase inducing complex medium was used in thecultivations. The spent culture medium obtained from the cultivationswas used in application tests (Examples 6-11) and as starting materialfor purification and further characterization of the recombinantFa_RF7182 protease.

Example 5 Purification and Characterization of the Recombinant Fa_RF7182Protease

Cells and solids were removed from the spent culture medium obtainedfrom the fermentation (Example 4) by centrifugation for 30 min, 50000 gat +4° C. (Sorvall RC6 plus). 15 ml of the supernatant was used forpurification of protease. All purification steps were performed at coldroom. After centrifugation, sample was filtered through 0.44 μm filter(MILLEX HV Millipore) before applying to HiPrep 26/10 Desalting column(from GE Healthcare) equilibrated in 20 mM Tris pH 8.5. Gel filteredsample was applied to a 20 mL Q Sepharose FF column (from GE Healthcare)equilibrated in 20 mM Tris pH 8.5. The flow through fraction wascollected and analysed on 12% SDS PAGE gel (FIG. 3). This enzyme samplewas used for characterization of pH and temperature profiles.

Temperature Profile

Temperature profile was obtained for Fa_RF7182 protease by using theassay described in Example 1c, except using 15 min reaction time at pH9. The result is shown in FIG. 4A. The protease has an optimaltemperature around 50° C.

pH-Profile

The pH profile of the protease was determined at 50° C. using casein asa substrate as described in Example 1c, except that casein was dissolvedin 40 mM Britton-Robinson buffer, the pH of the reaction was adjusted topH 6-12, the reaction time was 15 min and the enzyme reactions werestopped using a 0.11 M TCA solution which contained 0.22 M sodiumacetate and 0.33 M acetic acid. The results are shown in FIG. 4B. Therecombinant Fa_RF7182 protease exhibits relative activity over 85% frompH 6 to pH 9. The pH profile of the purified recombinant Fa_RF7182protease corresponded to the pH profile of the wild type Fa_RF7182protease purified as described in Example 1a.

Example 6 Performance of Recombinant Protein Fa_RF7182 at pH 9 Buffer atDifferent Temperatures

Recombinant protein Fa_RF7182 preparation produced in Trichoderma (asdescribed in Example 4), was tested for its ability to removeblood/milk/ink standard stain (Art.116, 100% cotton, EMPA TestmaterialenAG, Swizerland) at temperatures 30° C. and 50° C. Commercial proteasepreparations Savinase® Ultra 16 L (Novozymes) and Purafect® 4000 L(Genencor International) and treatment without enzyme (control) wereused for comparison. The stain fabric was first cut in to 1.5 cm×1.5 cmswatches and the pieces were made rounder by cutting the corners. Pieceswere placed in wells of microtiter plates (Nunc 150200). Into each wellhaving diameter of 2 cm, 1.5 ml enzyme dilution in Glysine-NaOH bufferpH 9 was added on top of the fabric. Each enzyme was dosed 0, 0.2, 0.4,0.8, 1.6, 4, and 8 activity units (μmol tyrosine/min) per 1.5 ml buffer.Activity was measured using 30 min reaction time as described in Example1(c) and using 10 min time for color development after addition ofdiluted Folin reagent. Microtiter plates with samples were incubated ina horizontal shaker at 30° C. and 50° C. for 60 min with 125 rpm. Afterthat the swatches were carefully rinsed under running water (appr. 45°C.) and dried overnight at indoor air on a grid, protected againstdaylight.

The stain removal effect was evaluated by measuring the colour asreflectance values Minolta CM 2500 spectrophotometer using L*a*b* colourspace coordinates (illuminant D65/2°.) The colour from both sides of theswatches was measured after the treatment. Each value was the average ofat least 2 parallel fabric samples measured from both side of thefabric. Fading of blood/milk/ink stain indicating of the proteaseperformance (stain removal efficiency) was calculated as ΔL*, whichmeans lightness value L* of enzyme treated fabric minus lightness valueL* of fabric treated with washing liquor (buffer) without enzyme (enzymeblank, control).

The results are shown in FIGS. 5 and 6. Fa_RF7182 protease preparationshowed considerably higher stain removal capacity especially at 30° C.in pH 9 buffer compared to commercial protease preparations Savinase®Ultra 16L and Purafect® 4000L.

Example 7 Performance of Recombinant Protein Fa_RF7182 with DetergentPowder at 40° C. And pH 10

Recombinant protein Fa_RF7182 preparation produced Trichoderma (asdescribed in Example 4) was tested for its ability to removeblood/milk/ink standard stain in the presence of phosphate containingreference detergent at 40° C. (pH ca. 10). Standard stain Art.117(blood/milk/ink, polyester+cotton, EMPA) was used as test material.Commercial protease Purafect® 4000L and treatment without enzyme(control) were used for comparison. Each enzyme was dosed 0, 0.2, 0.4,0.8, 1.6, 4, and 8 activity units (μmol tyrosine/min) per ml washliquor. Activity was measured as described in Example 6.

An amount of 3.3 g of phosphate containing ECE reference detergent 77without optical brightener (Art. 601, EMPA) was dissolved in 1 liter oftap water (dH≦4), mixed well with magnetic stirrer and tempered to 40°C. Stain fabric was cut into pieces like described in Example 6.Swatches were placed in well's of microtiter plates (Nunc 150200) and1.5 ml wash liquor containing detergent and enzyme dilution in water(below 70 μl) was added on top of the fabric. The plates with sampleswere in incubated in horizontal shaker at 40° C. for 60 min with 125rpm. After that the swatches were carefully rinsed under running water(appr. 45° C.) and dried overnight at indoor air, on a grid, protectedagainst daylight.

The colour of the swatches after treatment was measured with Minolta CM2500 spectrophotometer using L*a*b* colour space coordinates stainremoval effect calculated as ΔL* as described in example 6.

The results (FIG. 7) showed that protease Fa_RF7182 is also suitablewith for powder detergents at very alkaline conditions. The performancewas similar to commercial protease Purafect® 4000L at 40° C.

Example 8 Performance of Recombinant Protein Fa_RF7182 with LiquidDetergent at 40° C.

Recombinant protein Fa_RF7182 preparation produced in Trichoderma (asdescribed in Example 4) was tested for its ability to removeblood/milk/ink standard stain in the presence of liquid detergent ArielSensitive (Procter & Gamble, England), not containing enzyme, at 40° C.and pH ca. 7.9. Standard stain, artificially soiled test cloth Art.117(blood/milk/ink, polyester+cotton, EMPA) was used as test material.Commercial protease preparations Purafect® 4000L and Savinase Ultra® 16L and treatment without enzyme (control) were used for comparison. Eachenzyme was dosed 0, 0.2, 0.4, 0.8, 1.6, 4, and 8 activity units (μmoltyrosine/min) per ml wash liquor. Activity was measured as described inExample 6.

An amount of 3.3 g of Ariel Sensitive was dissolved in 1 liter of tapwater (dH≦4), mixed well with magnetic stirrer and temperated to 40° C.Stain fabric was cut into pieces like described in Example 6. Swatcheswere placed in well's of microtiter plates (Nunc 150200) and 1.5 ml washliquor containing detergent and enzyme dilution in water (<70 μl) wasadded on top of the fabric. The plates with samples were in incubated ina horizontal shaker at 40° C. for 60 min with 125 rpm. After that theswatches were carefully rinsed under running (appr. 45° C.) water anddried overnight at indoor air, on a grid, protected against daylight.

The colour of the swatches after treatment was measured with MinoltaCM2500 spectrophotometer using L*a*b* color space coordinates and stainremoval effect calculated as ΔL* as described in example 6.

Based on the results shown in FIG. 8 Fa_RF7182 has excellent performancewith liquid detergent at 40° C. The efficiency of Fa_RF7182 onblood/milk/ink/stain (polyester+cotton) was considerably higher comparedto commercial preparations Purafect® 4000L and Savinase® Ultra 14 L,when same amount of protease activity was dosed. The dosage of 4-8 unitsof commercial enzymes per ml of liquor was equal to dosage about0.2-0.5% of enzyme preparation per weight of detergent, which is intypical use level for detergent enzymes.

Example 9 Performance of Recombinant Protein Fa_RF7182 with DifferentLiquid Detergent Concentrations at 30° C.

Recombinant protein Fa_RF7182 preparation produced in Trichoderma (asdescribed in Example 4) was tested for its ability to removeblood/milk/ink standard stain with liquid detergent at concentrations1-5 g/l at 30° C. Ariel Sensitive (Procter & Gamble, England) containingno enzymes and liquid base detergent for coloured fabric containing 25%washing active substances, polyol and polymers (Table 4) were used asdetergents and standard stain Art.117 (blood/milk/ink, cotton+polyester,EMPA) was used as test material. Commercial protease preparationsPurafect® 4000L, Savinase® Ultra 16 L and treatment without enzyme(control) were used for comparison. Each enzyme was dosed 0, 0.2, 0.4,0.8, 1.6, 4, and 8 activity units (μmol tyrosine/min) per ml washliquor. Activity was measured as described in Example 6.

TABLE 4 Composition of Liquid Base detergent for colored fabricIngredients % NaLES (sodium lauryl ether sulphate) 4.9 Nonionic C12-157EO (ethylene oxide) 15 Na-Soap (Palm Kernel FA) 4.4 Coco Glucoside 1<Total Surfactant> <25.30> Polyol (Glycerin) 5 Phosphonate (32%)(ThermPhos) 2 PVP-Sokalan HP 53 (BASF) 1 Sokalan PA 15 (BASF) 1.56Sorez-100 (ISP) 0.4 Water up to 100%

Amounts of 1, 3.3 and 5 g of liquid detergent was dissolved in 1 literof tap water (dH≦4), mixed well with magnetic stirrer and tempered to30° C. The pH in the wash liquors was ca. 7.3-7.5 with Base detergent orca. 7.6-8.0 with Ariel, depending on detergent concentration. Stainfabric was cut into pieces like described in Example 6. Swatches wereplaced in wells of microtiter plates (Nunc 150200) and 1.5 ml washliquor containing detergent and enzyme dilution in water (below 70 μl)was added on top of the fabric. The plates with samples were inincubated in a horizontal shaker at 30° C. for 60 min with 125 rpm.After that the swatches were carefully/thoroughly rinsed under warmrunning water and dried overnight at indoor air, on a grid, protectedagainst daylight.

The colour of the swatches after treatment was measured with Minolta CM2500 spectrophotometer using L*a*b* color space coordinates and stainremoval effect calculated as ΔL* as described in example 6.

Results obtained with base detergent for coloured fabrics are shown inFIGS. 9 A-D and results obtained with Ariel Sensitive are shown in FIG.10 A-D. The efficiency of Fa_RF7182 on blood/milk/ink stain wasconsiderably higher compared to commercial preparations Purafect® 4000Land Savinase® Ultra 14 L with both detergents and at all detergentconcentrations, when same amount of activity was dosed. Also if dosingis calculated as amount of added protein (FIGS. 9B and 10B), the stainremoval efficiency is highest with Fa_RF7182. The amount of protein fromthe enzyme preparations was determined by Bio-Rad protein assay (Bio-RadLaboratories, Hercules, Calif.) using bovine gammaglobulin (Bio-Rad) asstandard. Results of these tests indicate that Fa_RF7182 protease hasexcellent performance with liquid detergents at low temperatures, like30° C.

Example 10 Efficiency of Recombinant Protein Fa_RF7182 on DifferentStains with Liquid Detergent at 30° C. (Launder Ometer)

Recombinant protein Fa_RF7182 preparation produced Trichoderma (asdescribed in Example 4) was tested for its ability to remove differentstains with liquid detergent at 30° C. and compared to commercialprotease preparations Purafect® 4000L and/or Savinase® Ultra 16 L. Thefollowing artificially soiled test cloths from EMPA were used:blood/milk/ink Art.117 (polyester+cotton), blood/milk/ink Art.116(cotton), grass Art. 164 and cocoa Art.112. The fabric was cut in 9cm×12 cm swatches and the edges were neated by zig-zag stitches.Detergent was the same as described in example 9.

Stain removal treatments were performed in LP-2 Launder Ometer asfollows. Launder Ometer was first preheated to 30° C. 450 ml of temperedwash liquor containing 5 g detergent per liter tap water (dH≦4) andenzyme dilution was added in containers containing stains Art. 116 and117 or stains Art. 164 and Art. 112. Enzymes were dosed 0, 1 and/or 2,5, 10 activity units (μmol tyrosine/min) per ml wash liquor withblood/milk/ink stains and 0, 5, 10 and 20 activity units (μmoltyrosine/min) per ml wash liquor with grass and cocoa. Activity wasmeasured as described in Example 6. The Launder Ometer was run at 30° C.for 60 min ca. pH 7.5. After that the swatches were carefully rinsedunder running water (ca. 20° C.) and dried overnight at indoor air, on agrid, protected against daylight.

The colour of the swatches after treatment was measured with Minolta CM2500 spectrophotometer using L*a*b* color space coordinates and stainremoval effect calculated as ΔL* as described in example 6. Each valuewas the average of at least 20 measurements per swatch. The measurementswere avoided from areas with crease marks formed during the treatmentbecause of the folding of the fabric (in cotton stains Art. 116 and Art.112).

Results obtained with base detergent (5 g/l) for coloured fabrics (FIGS.11 A-D) showed that efficiency on blood/milk/ink, grass and cocoa stainswas considerably higher with Fa_RF7182 compared to commercialpreparations Savinase® Ultra 16L and Purafect® 4000 L at 30° C. whensame amount of protease activity was dosed. The dosage of 10 units ofcommercial enzymes per ml of was liquor was equal to dosageapproximately 0.4% of enzyme preparation per weight of detergent, whichis in typical use level for detergent enzymes. Results of these testsindicate that Fa_RF7182 protease is efficient on several stains at lowtemperatures like 30° C.

Example 11 Evaluation of the Performance of the Recombinant ProteinFa_RF7182 in Liquid Laundry Detergent 30° C. in Full Scale Trials

The performance of recombinant protein Fa_RF7182 preparation producedTrichoderma (as described in Example 4) was tested in liquid detergentin full scale in a washing machine at 30° C. and compared to commercialprotease preparations Purafect® 4000L and Savinase® Ultra 16L andtreatment with detergent without enzyme. Liquid base detergent forcoloured fabrics, as described in Example 9, and 8 different proteasesensitive tracers (Table 5) were used. In addition two pieces of ballastsoil (CFT-SBL) per wash were placed in wash net to avoid contaminationby contact with the other swatches. Tracers were from CFT (Center ForTestmaterials BV, The Netherlands). Stain swatches 10 cm×10 cm werestitched to kitchen towels. The process parameters and conditions aredescribed in Table 6. Enzyme dosages used in the trials were calculatedboth as enzyme activities (ca. 0-14 activity units per ml wash liquor)and as amount of protein (ca. 0-2.5 mg per liter wash liquor). Proteaseactivities and protein contents of the preparations were measured asdescribed in Examples 6 and 9.

TABLE 5 Protease sensitive tracers used in test. Nr. Monitor/SwatchSubstrate 1 CFT/CS-01-106 Blood (aged)/Cotton 2 CFT/C-03-030 Chocolatemilk/pigment/Cotton 3 CFT/C-05-059b Bood/milk/ink/Cotton 4 CFT/PC-05-014Blood/Milk/Ink/PE-Cotton 5 CFT/CS-08-069 Grass/Cotton 6 CFT/C-10-186bGroundnut oil/milk/Cotton 7 CFT/CS- 25-016 Spinach/Cotton 8CFT/CS-38-010 Egg Yolk/Pigment/Cotton

TABLE 6 Process parameters and conditions Machine Miele N-TronicFrontstar Program 30° C., short program Hardness of water about 11.2° dHwith 13 liter intake Ballast Load 2.5 kg bed sheets/bath towels, andkitchen towels Detergent dosage 80 g/machine load Amount of each tracers2 stain trips 10 cm × 10 cm per machine Number of washes 2

Evaluation of stain removal effect was based on measurement ofreflectance (remission), R 457 nm, with Datacolor-spectrophotometer withan UV-filter and calculated as percentage stain removal effect (% SR):

${\%{SR}} = \frac{\begin{matrix}{{{Reflectance}\mspace{14mu}{after}\mspace{14mu}{washing}} -} \\{{Reflectance}\mspace{14mu}{before}\mspace{14mu}{washing} \times 100\%}\end{matrix}}{{{Reflectance}\mspace{14mu}{of}\mspace{14mu}{unsoiled}\mspace{14mu}{fabric}} - {{Reflectance}\mspace{14mu}{before}\mspace{14mu}{washing}}}$

Results were calculated as Δ% SR (delta % SR), which means % SR ofenzyme treated fabric minus % SR of treatment without enzyme (detergentonly).

Two washes containing two swatches of each stain were performed witheach dosage of enzyme and three measurements were measured of each stainswatch, so the values are based upon the 12 measurements per stain perproduct.

The results of total soil removal efficiencies (delta % SR) are shown inFIGS. 12A-B and FIGS. 13 A-E. The total stain removal effect based onthe sum of the results obtained with the eight different proteasesensitive stains (Stains 1-8, Table 5) was considerably higher withFa_RF7182 compared to commercial protease preparations Purafect® 4000Land Savinase® Ultra 16L, when proteases were dosed as amount of activity(FIG. 12A). Fa_RF7182 was efficient especially on blood/milk/ink,chocolate/milk, groundnut oil/milk and egg yolk (FIGS. 13A-E).

A general detergency tracer (pigment/oil) was used as a control inrepeats in different machines. No differences between the various testswere observed.

Example 12 Performance of Recombinant Protein Fa_RF7182 in pH 9 Bufferat Temperatures from 10° C. to 60° C.

Recombinant protein Fa_RF7182 produced in Trichoderma (as described inExample 4) was tested for its ability to remove blood/milk/ink standardstain (Art.117, polyester+cotton, EMPA Testmaterialen AG, Swizerland) attemperatures from 10° C. to 60° C. Commercial protease preparationsSavinase® Ultra 16 L, Purafect® 4000 L and Properase® 4000 E andtreatment without enzyme (control) were used for comparison. The testswere performed at pH 9 buffer as described in Example 6, except theincubation temperature range was broader, stain material was differentand the temperature of the rinsing water of the swatches was similar towashing temperature. The colour of the swatches after treatment wasmeasured with Minolta CM 2500 spectrophotometer using L*a*b* color spacecoordinates and stain removal effect calculated as ΔL* as described inExample 6.

The results are shown in FIGS. 14A-F. Fa_RF7182 protease preparationshowed considerably higher stain removal capacity especially at thelowest temperatures from 10° C. to 40° C. in pH 9 buffer, compared tocommercial protease preparations Savinase® Ultra 16L and Purafect® 4000Land Properase® 4000 E.

Example 13 Performance of Recombinant Protein Fa_RF7182 with LiquidDetergent at Cold Washing Temperatures (10° C. And 20° C.)

Recombinant protein Fa_RF7182 produced in Trichoderma (as described inExample 2) was tested for its ability to remove blood/milk/ink standardstain (Art.117, cotton+polyester, EMPA) with Liquid Base detergent atlow temperatures. The testing method was similar to Example 9, exceptonly detergent concentration of 3.3 g/l and lower incubationtemperatures, 10° C. and 20° C., were used. The temperature of therinsing water of the swatches was similar to washing temperature.

The colour of the swatches after treatment was measured with Minolta CM2500 spectrophotometer using L*a*b* color space coordinates and stainremoval effect calculated as ΔL* as described in Example 6. Fortreatment without enzyme (enzyme blank), detergent solution was used aswashing liquor.

Results obtained with Liquid Base detergent concentration of 3.3 g/l at10° C. and 20° C. are shown in FIGS. 15 A and B. The efficiency ofFa_RF7182 on blood/milk/ink stain was considerably higher both at 10° C.and 20° C. compared to commercial preparations Savinase® Ultra 16L,Purafect® 4000L and Properase® 4000E, when same amount of activity wasdosed. Results of these tests indicate that Fa_RF7182 protease hasexcellent performance with liquid detergents at very low washingtemperatures.

REFERENCES

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The invention claimed is:
 1. An expression vector, comprising a nucleicacid sequence selected from the group consisting of: (a) a nucleic acidsequence encoding a polypeptide exhibiting the serine proteasespecificity of SEQ ID NO:18 and comprising the amino acid sequence setforth in SEQ ID NO:18; (b) a nucleic acid sequence encoding apolypeptide that comprises an amino acid sequence that exhibits serineprotease activity and is at least 92% identical to the amino acidsequence set forth in SEQ ID NO:18, wherein sequence identity isdetermined using ClustalW alignment using matrix: BLOSUM, Gap Open: 10,and Gap Extension: 0.5, and wherein the amino acid sequence thatexhibits serine protease activity has the serine protease specificity ofSEQ ID NO:18; (c) a cDNA sequence comprising the coding sequence of thenucleotide sequence set forth in SEQ ID NO:13, wherein the polypeptideencoded by the coding sequence, in its mature form, exhibits the serineprotease specificity of SEQ ID NO:18; (d) a cDNA sequence comprising thecoding sequence of the nucleotide sequence set forth in SEQ ID NO:15,wherein the polypeptide encoded by the coding sequence, in its matureform, exhibits the serine protease specificity of SEQ ID NO:18; (e) acDNA sequence comprising the coding sequence of the nucleotide sequenceset forth in SEQ ID NO:17, wherein the polypeptide encoded by the codingsequence exhibits the serine protease specificity of SEQ ID NO:18; (f) acDNA sequence comprising the coding sequence of the nucleotide sequencein plasmid pALK2531 contained in the E. coli strain DSM 22209, whereinthe coding sequence encodes a polypeptide, which in its mature form,exhibits the serine protease specificity of SEQ ID NO:18; (g) a nucleicacid sequence the coding sequence of which differs from the codingsequence of the nucleotide sequence of any one of (c) to (f) due to thedegeneracy of the genetic code; (h) a nucleic acid sequence comprisingthe nucleotide sequence set forth in SEQ ID NO:15 fused at its 5′ end tothe nucleotide sequence of a heterologous signal sequence, SEQ ID NO:15encoding a polypeptide, which in its mature form, exhibits serineprotease activity; and (i) a nucleic acid sequence that hybridizes understringent conditions with the complement of the nucleic acid sequenceset forth in SEQ ID NO:12 contained in DSM 22208 and encodes apolypeptide comprising an amino acid sequence that exhibits the serineprotease specificity of SEQ ID NO:18 and is at least 92% identical tothe full-length amino acid sequence set forth in SEQ ID NO:18, whereinsequence identity is determined using ClustalW alignment using matrix:BLOSUM, Gap Open: 10, and Gap Extension: 0.5, and wherein stringentconditions comprise hybridization in a solution comprising 6×SSC,5×Denhardt's reagent, and 0.5% SDS at 65° C., and washing twice for 15minutes at 65° C. in a solution comprising 0.1×SSC and 0.1% SDS.
 2. Anucleic acid molecule, comprising a nucleic acid sequence selected fromthe group consisting of: (a) a cDNA sequence comprising the codingsequence of the nucleotide sequence set forth in SEQ ID NO:13, whereinthe polypeptide encoded by the coding sequence, in its mature form,exhibits serine protease activity; (b) a cDNA sequence comprising thecoding sequence of the nucleotide sequence set forth in SEQ ID NO:15,wherein the polypeptide encoded by the coding sequence, in its matureform, exhibits the serine protease specificity of SEQ ID NO:18; (c) acDNA sequence comprising the coding sequence of the nucleotide sequenceset forth in SEQ ID NO:17, wherein the polypeptide encoded by the codingsequence exhibits the serine protease specificity of SEQ ID NO:18; (d) acDNA sequence comprising the coding sequence of the nucleotide sequenceset forth in SEQ ID NO:13 in plasmid pALK2531 contained in the E. colistrain DSM 22209, wherein the coding sequence encodes a polypeptide,which in its mature form, exhibits the serine protease specificity ofSEQ ID NO:18; (e) a cDNA sequence the coding sequence of which differsfrom the coding sequence of the nucleotide sequence of any one of (a) to(d) due to the degeneracy of the genetic code; and (f) a cDNA sequencecomprising the coding sequence of the nucleotide sequence set forth inSEQ ID NO:15 fused at its 5′ end to the nucleotide sequence of aheterologous signal sequence, the coding sequence encoding apolypeptide, which in its mature form, exhibits the serine proteasespecificity of SEQ ID NO:18.
 3. The nucleic acid molecule of claim 2,wherein the nucleic acid sequence is a cDNA sequence comprising thecoding sequence of the nucleotide sequence set forth in SEQ ID NO:13,wherein the polypeptide encoded by the coding sequence, in its matureform, exhibits the serine protease specificity of SEQ ID NO:18.
 4. Thenucleic acid molecule of claim 2, wherein the nucleic acid sequence is acDNA sequence comprising the coding sequence of the nucleotide sequenceset forth in SEQ ID NO:15, wherein the polypeptide encoded by the codingsequence, in its mature form, exhibits the serine protease specificityof SEQ ID NO:18.
 5. The nucleic acid molecule of claim 2, wherein thenucleic acid sequence is a cDNA sequence comprising the coding sequenceof the nucleotide sequence set forth in SEQ ID NO:17, wherein thepolypeptide encoded by the coding sequence exhibits the serine proteasespecificity of SEQ ID NO:18.
 6. The nucleic acid molecule of claim 2,wherein the nucleic acid sequence is a cDNA sequence comprising thecoding sequence of the nucleotide sequence set forth in SEQ ID NO:13 inplasmid pALK2531 contained in E. coli strain DSM 22209, wherein thecoding sequence encodes a polypeptide, which in its mature form,exhibits the serine protease specificity of SEQ ID NO:18.
 7. The nucleicacid molecule of claim 2, wherein the nucleic acid sequence is a cDNAsequence comprising the coding sequence of the nucleotide sequence setforth in SEQ ID NO:15 fused at its 5′ end to the nucleotide sequence ofa heterologous signal sequence, the coding sequence encoding apolypeptide, which in its mature form, exhibits the serine proteasespecificity of SEQ ID NO:18.
 8. The expression vector of claim 1,wherein the vector comprises a nucleic acid sequence encoding apolypeptide exhibiting the serine protease specificity of SEQ ID NO:18and comprising the amino acid sequence set forth in SEQ ID NO:18, thenucleic acid sequence operably linked to regulatory sequences capable ofdirecting expression of the polypeptide encoded by the nucleic acid in ahost cell.
 9. An expression vector comprising the nucleic acid moleculeof claim 3 operably linked to regulatory sequences capable of directingexpression of the polypeptide encoded by the coding sequence in a hostcell.
 10. An expression vector comprising the nucleic acid molecule ofclaim 4 operably linked to regulatory sequences capable of directingexpression of the polypeptide encoded by the coding sequence in a hostcell.
 11. An expression vector comprising the nucleic acid molecule ofclaim 5 operably linked to regulatory sequences capable of directingexpression of the polypeptide encoded by the coding sequence in a hostcell.
 12. An expression vector comprising the nucleic acid molecule ofclaim 6 operably linked to regulatory sequences capable of directingexpression of the polypeptide encoded by the coding sequence in a hostcell.
 13. An expression vector comprising the nucleic acid molecule ofclaim 7 operably linked to regulatory sequences capable of directingexpression of the polypeptide encoded by the coding sequence in a hostcell.
 14. A host cell comprising the expression vector of claim
 8. 15. Ahost cell comprising the expression vector of claim
 9. 16. A host cellcomprising the expression vector of claim
 10. 17. A host cell comprisingthe expression vector of claim
 11. 18. A host cell comprising theexpression vector of claim
 12. 19. A host cell comprising the expressionvector of claim
 13. 20. The host cell of claim 17, wherein the host cellis a microbial host cell.
 21. The host cell of claim 17, wherein thehost cell is a cell from a filamentous fungus.
 22. The host cell ofclaim 21, wherein the filamentous fungus is of the genus Trichoderma,Aspergillus, Fusarium, Humicola, Chrysosporium, Neurospora, Rhizopus,Penicillium, or Mortierella.
 23. The host cell of claim 21, wherein thefilamentous fungus is of the genus Trichoderma.
 24. The host cell ofclaim 21, wherein the filamentous fungus is of the genus Aspergillus.25. The host cell of claim 23, wherein the host cell is from Trichodermareesei.
 26. The host cell of claim 17, wherein the host cell is aheterologous host cell.
 27. The expression vector of claim 1, whereinthe expression vector comprises the nucleic acid sequence encoding apolypeptide that comprises an amino acid sequence that exhibits serineprotease activity and is at least 92% identical to the amino acidsequence set forth in SEQ ID NO:18, wherein sequence identity isdetermined using ClustalW alignment using matrix: BLOSUM, Gap Open: 10,and Gap Extension: 0.5, and wherein the amino acid sequence thatexhibits serine protease activity has the serine protease specificity ofSEQ ID NO:18, the nucleic acid sequence operably linked to regulatorysequences capable of directing expression of the polypeptide encoded bythe nucleic acid in a host cell.
 28. The expression vector of claim 1,wherein the expression vector comprises the nucleic acid sequence thathybridizes under stringent conditions with the complement of the nucleicacid sequence set forth in SEQ ID NO:12 contained in DSM 22208 andencodes a polypeptide comprising an amino acid sequence that exhibitsthe serine protease specificity of SEQ ID NO:18 and is at least 92%identical to the full-length amino acid sequence set forth in SEQ IDNO:18, wherein sequence identity is determined using ClustalW alignmentusing matrix: BLOSUM, Gap Open: 10, and Gap Extension: 0.5, and whereinstringent conditions comprise hybridization in a solution comprising6×SSC, 5×Denhardt's reagent, and 0.5% SDS at 65° C., and washing twicefor 15 minutes at 65° C. in a solution comprising 0.1×SSC and 0.1% SDS,the nucleic acid sequence operably linked to regulatory sequencescapable of directing expression of the polypeptide encoded by thenucleic acid in a host cell.
 29. A host cell comprising the expressionvector of claim
 28. 30. The host cell of claim 29, wherein the host cellis a microbial host cell.
 31. The host cell of claim 29, wherein thehost cell is a cell from a filamentous fungus.
 32. The host cell ofclaim 31, wherein the filamentous fungus is of the genus Trichoderma,Aspergillus, Fusarium, Humicola, Chrysosporium, Neurospora, Rhizopus,Penicillium, or Mortierella.
 33. The host cell of claim 32, wherein thefilamentous fungus is of the genus Trichoderma.
 34. The host cell ofclaim 32, wherein the filamentous fungus is of the genus Aspergillus.35. A host cell comprising the expression vector of claim
 27. 36. Theexpression vector of claim 27, wherein the nucleic acid sequence encodesa polypeptide that comprises an amino acid sequence that exhibits serineprotease activity and is at least 94% identical to the amino acidsequence set forth in SEQ ID NO:18.
 37. The expression vector of claim27, wherein the nucleic acid sequence encodes a polypeptide thatcomprises an amino acid sequence that exhibits serine protease activityand is at least 96% identical to the amino acid sequence set forth inSEQ ID NO:18.
 38. The expression vector of claim 27, wherein the nucleicacid sequence encodes a polypeptide that comprises an amino acidsequence that exhibits serine protease activity and is at least 98%identical to the amino acid sequence set forth in SEQ ID NO:18.
 39. Theexpression vector of claim 27, wherein the nucleic acid sequence encodesa polypeptide that comprises an amino acid sequence that exhibits serineprotease activity and is at least 99% identical to the amino acidsequence set forth in SEQ ID NO:18.
 40. The host cell of claim 35,wherein the host cell is a cell from a filamentous fungus of the genusTrichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium, Neurospora,Rhizopus, Penicillium, or Mortierella.
 41. The host cell of claim 15,wherein the host cell is a microbial cell.
 42. The host cell of claim14, wherein the host cell is a microbial host cell.
 43. The host cell ofclaim 14, wherein the host cell is a cell from a filamentous fungus. 44.The host cell of claim 43, wherein the filamentous fungus is of thegenus Trichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium,Neurospora, Rhizopus, Penicillium, or Mortierella.
 45. The host cell ofclaim 44, wherein the filamentous fungus is of the genus Trichoderma.46. The host cell of claim 44, wherein the filamentous fungus is of thegenus Aspergillus.